Modulators of complement activity

ABSTRACT

The present invention provides polypeptide modulators of complement activity, including cyclic polypeptide modulators. Also provided are methods of utilizing such modulators as therapeutics.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. application Ser. No.15/547,085 filed Jul. 28, 2017, entitled Modulators of of ComplementActivity, which is a 35 U.S.C. § 371 U.S. National Stage Entry ofInternational Application No. PCT/US2016/015412 filed Jan. 28, 2016,Modulators of of Complement Activity, which claims the benefit of U.S.Provisional Patent Application No. 62/108,772 filed Jan. 28, 2015,entitled Modulation of Complement Activity, International PublicationNo. PCT/US2015/035473 filed Jun. 12, 2015, entitled Modulation ofComplement Activity, and U.S. Provisional Patent Application No.62/185,298 filed Jun. 26, 2015, entitled Modulation of ComplementActivity, the contents of each of which are herein incorporated byreference in their entirety.

SEQUENCE LISTING

The instant application contains a Sequence Listing which has beensubmitted electronically in ASCII format and is hereby incorporated byreference in its entirety. Said ASCII copy, created on Feb. 26, 2018, isnamed 2011_1007USCON_SL.txt and is 126,131 bytes in size.

FIELD OF THE INVENTION

The present invention relates to compounds, including polypeptides,which are useful as modulators of complement activity. Also provided aremethods of utilizing these modulators as therapeutics.

BACKGROUND OF THE INVENTION

The vertebrate immune response is comprised of adaptive and innateimmunity components. While the adaptive immune response is selective forparticular pathogens and is slow to respond, components of the innateimmune response recognize a broad range of pathogens and respond rapidlyupon infection. One such component of the innate immune response is thecomplement system.

The complement system includes about 20 circulating proteins,synthesized primarily by the liver. Components of this particular immuneresponse were first termed “complement” due to the observation that theycomplemented the antibody response in the destruction of bacteria. Theseproteins remain in an inactive form prior to activation in response toinfection. Activation occurs by way of a pathway of proteolytic cleavageinitiated by pathogen recognition and leading to pathogen destruction.Three such pathways are known in the complement system and are referredto as the classical pathway, the lectin pathway and the alternativepathway. The classical pathway is activated when an IgG or IgM moleculebinds to the surface of a pathogen. The lectin pathway is initiated bythe mannan-binding lectin protein recognizing the sugar residues of abacterial cell wall. The alternative pathway remains active at lowlevels in the absence of any specific stimuli. While all three pathwaysdiffer with regard to initiating events, all three pathways convergewith the cleavage of complement component C3. C3 is cleaved into twoproducts termed C3a and C3b. Of these, C3b becomes covalently linked tothe pathogen surface while C3a acts as a diffusible signal to promoteinflammation and recruit circulating immune cells. Surface-associatedC3b forms a complex with other components to initiate a cascade ofreactions among the later components of the complement system. Due tothe requirement for surface attachment, complement activity remainslocalized and minimizes destruction to non-target cells.

Pathogen-associated C3b facilitates pathogen destruction in two ways. Inone pathway, C3b is recognized directly by phagocytic cells and leads toengulfment of the pathogen. In the second pathway, pathogen-associatedC3b initiates the formation of the membrane attack complex (MAC). In thefirst step, C3b complexes with other complement components to form theC5-convertase complex. Depending on the initial complement activationpathway, the components of this complex may differ. C5-convertase formedas the result of the classical complement pathway comprises C4b and C2ain addition to C3b. When formed by the alternative pathway,C5-convertase comprises two subunits of C3b as well as one Bb component.

Complement component C5 is cleaved by either C5-convertase complex intoC5a and C5b. C5a, much like C3a, diffuses into the circulation andpromotes inflammation, acting as a chemoattractant for inflammatorycells. C5b remains attached to the cell surface where it triggers theformation of the MAC through interactions with C6, C7, C8 and C9. TheMAC is a hydrophilic pore that spans the membrane and promotes the freeflow of fluid into and out of the cell, thereby destroying it.

An important component of all immune activity is the ability of theimmune system to distinguish between self and non-self cells. Pathologyarises when the immune system is unable to make this distinction. In thecase of the complement system, vertebrate cells express proteins thatprotect them from the effects of the complement cascade. This ensuresthat targets of the complement system are limited to pathogenic cells.Many complement-related disorders and diseases are associated withabnormal destruction of self cells by the complement cascade. In oneexample, subjects suffering from paroxysmal nocturnal hemoglobinuria(PNH) are unable to synthesize functional versions of the complementregulatory proteins CD55 and CD59 on hematopoietic stem cells. Thisresults in complement-mediated hemolysis and a variety of downstreamcomplications. Other complement-related disorders and diseases include,but are not limited to autoimmune diseases and disorders, neurologicaldiseases and disorders, blood diseases and disorders and infectiousdiseases and disorders. Experimental evidence suggests that manycomplement-related disorders are alleviated through inhibition ofcomplement activity. Therefore, there is a need for the development ofcompounds and methods for selectively blocking complement-mediated celldestruction and for treating related indications. The present inventionmeets this need by providing polypeptides described herein and relatedmethods for their use.

SUMMARY OF THE INVENTION

In some embodiments, the present invention provides a method of reducingthe cleavage of C5 in a biological system by at least 50% relative tocleavage of C5 in an untreated control system, comprising providing apolypeptide with the amino acid sequence of SEQ ID NO: 184 to thebiological system, wherein the polypeptide is provided at a finalconcentration of from about 0.1 nM to about 50 nM, and wherein cleavageof C5 is reduced by at least 50% relative to cleavage of C5 in theuntreated control system.

In some embodiments, the present invention provides a method of reducinghemolysis in a biological system by at least 50% relative to hemolysisin an untreated control system, comprising providing a polypeptide withthe amino acid sequence of SEQ ID NO: 184 to the biological system,wherein the polypeptide is provided at a final concentration of fromabout 0.1 nM to about 50 nM, and wherein hemolysis is reduced by atleast 50% relative to hemolysis in an untreated control system.

Also provided herein is a method of reducing hemolysis in a subject byabout 50% to about 95% relative to hemolysis levels previously observedin a subject, comprising administering a polypeptide with the amino acidsequence of SEQ ID NO: 184, wherein the polypeptide is administered at adose sufficient to achieve plasma levels of the polypeptide of fromabout 0.2 mg/L (0.1 μM) to about 40 mg/L (20 μM). Such subjects may behuman, non-human primate, or porcine subjects. In some cases, subjectsare human subjects with paroxysmal nocturnal hemoglobinuria (PNH).

In some embodiments, polypeptides with the amino acid sequence of SEQ IDNO: 184 are administered to human subjects at a dose of about 0.01 mg/kgto about 20 mg/kg. Such administration may be carried out daily orweekly.

In some embodiments, the present invention provides a method of reducinghemolysis in a subject with PNH comprising administering a polypeptidewith the amino acid sequence of SEQ ID NO: 184, wherein the polypeptideis administered at a dose of from about 0.01 mg/kg to about 20 mg/kg andwherein administration is daily, weekly or monthly. In some cases, suchsubjects have been treated previously or are currently receivingtreatment with ECULIZUMAB®.

Also provided is a method of reducing hemolysis in a subject comprisingadministering a polypeptide, wherein the subject has PNH, and whereinthe subject is not responsive to treatment with ECULIZUMAB®.

According to some methods of the invention, hemolysis is reduced byabout 50% to about 95% relative hemolysis observed previously in asubject.

DESCRIPTION OF THE FIGURES

The foregoing and other objects, features and advantages will beapparent from the following description of particular embodiments of theinvention, as illustrated in the accompanying drawings in which likereference characters refer to the same parts throughout the differentviews. The drawings are not necessarily to scale, emphasis instead beingplaced upon illustrating the principles of various embodiments of theinvention.

FIG. 1 is a line graph displaying the results of an enzyme immunoassay(EIA) for the detection of C5a in supernatant from a human red bloodcell (RBC) hemolysis assay with increasing concentrations of inhibitorsR3002 (SEQ ID NO: 3) and R3008 (SEQ ID NO: 9). Levels of C5a correlatewith complement activity and are therefore an indicator of the abilityof the compounds tested to inhibit complement activity. Supernatant fromthe hemolysis assay was diluted 1:50 and assayed for C5a levels. C5alevels decreased in human hemolysis assay supernatant samples withincreasing levels of either inhibitor assayed. R3002 (SEQ ID NO: 3) hadan IC₅₀ of 5.4 nM while R3008 (SEQ ID NO: 9) had an IC₅₀ of 54.5 nM. Asused herein, the term “IC₅₀” refers to the half maximal inhibitoryconcentration, a value used to indicate the amount of the inhibitorneeded to reduce a given reaction or process by half.

FIG. 2 is a line graph displaying the results of an EIA for thedetection of the membrane attack complex (MAC) in supernatant from ahuman RBC hemolysis assay with increasing concentrations of R3008 (SEQID NO: 9). Levels of the MAC correlate with complement activity and aretherefore an indicator of the ability of R3008 (SEQ ID NO: 9) to inhibitcomplement activity. Supernatant from the hemolysis assay was diluted1:5 and assayed for MAC levels. MAC levels decreased in hemolysis assaysupernatant samples with increasing levels of the inhibitor assayed withan IC₅₀ of 33 nM.

FIG. 3 is a line graph displaying competitive fluorescence polarization(FP) data for test articles R3003 (SEQ ID NO: 4), R3011 (SEQ ID NO: 31),R3014 (SEQ ID NO: 55), R3023 (SEQ ID NO: 104), R3043 (SEQ ID NO: 50) andR3050 (SEQ ID NO: 23). FP allows binding events to be measured in ahomogenous solution. A competitive binding assay was conducted wherein a25 nM solution of compound R3076 (SEQ ID NO: 40), which has afluorescent tag, was combined with increasing amounts of the testarticles and measured for changes in FP (in milli-polarization units;mP). Decreasing mP levels correlate with successful competition for C5by the test articles. The averages of two independent experimentsconducted in triplicate (+/−standard deviation) are shown. Of thearticles tested, R3003 (SEQ ID NO: 4) was the most potent while R3023(SEQ ID NO: 104), a control polypeptide, showed no activity at thehighest concentration tested.

FIG. 4 is a line graph showing results from a study in cynomolgusmonkey. Changes in R3152 (SEQ ID NO: 153) plasma concentration (circles)following a single 3 mg/kg IV dose in cynomolgus monkey are shown. Alsoshown are changes in hemolytic activity (squares) at the same timepoints.

FIG. 5 is a line graph showing results of compound monitoring in plasmafollowing intravenous (IV; squares) or subcutaneous (SC; circles)administration of 2 mg/kg of R3152 (SEQ ID NO: 153) in maleSprague-Dawley rats. Monitoring comprised determination of combinedplasma concentrations of R3152 (SEQ ID NO: 153) as well as itsequipotent C-terminally deamidated metabolite, R3201 (SEQ ID NO: 211.)

FIGS. 6A and 6B are line graphs depicting the pharmacokinetics ofcompounds of the present invention in rats. Male Sprague-Dawley rats(n=3) were injected intravenously at a single 2 mg/kg dose. Bloodsamples were collected at indicated time points, processed into plasma,and analyzed for the indicated compound by LC-MS (FIG. 6A). Blackcircles indicate results with R3176 (SEQ ID NO: 177) (unlipidatedcompound) and open circles indicate results with R3183 (SEQ ID NO: 184)(C16 lipidated compound). Male Sprague-Dawley rats (n=3) were alsoinjected subcutaneously at a single 15 mg/kg dose. Blood samples werecollected at indicated time points, processed into plasma, and analyzedfor the indicated compound by LC-MS (FIG. 6B). Black circles indicateresults with R3176 (SEQ ID NO: 177) (unlipidated compound) and opencircles indicate results with R3183 (SEQ ID NO: 184) (C16 lipidatedcompound).

FIG. 7 is a scatter plot presenting the effects of R3183 (SEQ ID NO:184) (C16 lipidated compound) or an anti-C5 monoclonal antibody similarto ECULIZUMAB® on inhibition of hemolysis via the thrombin-inducedcomplement pathway.

FIG. 8 is a line graph showing results from surface plasmon resonanceanalysis of C5 binding by R3183 (SEQ ID NO: 184).

FIG. 9 is a line graph showing results of a C5a immunoassay with humanred blood cell hemolysis assay supernatant.

FIG. 10 is a line graph showing results of a membrane attack complex(MAC) formation immunoassay performed on supernatant from a human redblood cell hemolysis assay.

FIGS. 11A and 11B are line graphs showing results of human hemolysisassays. FIG. 11A is a line graph comparing inhibition betweenECULIZUMAB® and R3183 (SEQ ID NO: 184). FIG. 11B is a line graphpresenting results of a hemolysis assay with R3183 (SEQ ID NO: 184) inthe presence of human or non-human primate serum.

FIGS. 12A and B are line graphs showing results from compoundadministration in animal models. FIG. 12A is a line graph showingresults of R3183 administration in an animal model. FIG. 12B is a linegraph showing results of R3183 administration in an animal model.

FIG. 13 is a scatter plot showing combined data from pharmacokinetic andpharmacodynamics studies.

FIG. 14 is a bar graph showing results of a hemolysis assay.

DETAILED DESCRIPTION

The present invention relates to the discovery of novel C5 modulatorycompounds and methods of their use. Such compounds may include, but arenot limited to polypeptides (e.g. cyclic polypeptides, peptidomimeticsand cyclic peptidomimetics), small molecules, antibodies, antibodyfragments, and aptamers. In some cases, C5 modulatory compounds arepolypeptides useful in the diagnosis and/or treatment of diseases inwhich the inhibition of complement activation is desirable. In someembodiments, polypeptides of the invention specifically bind complementcomponent C5. In further embodiments, polypeptides of the inventionreduce complement-mediated cell lysis (e.g., red blood cell hemolysis)by preventing cleavage of C5 into C5a and C5b fragments.

Definitions

Biological system: As used herein, the term “biological system” refersto a cell, a group of cells, a tissue, an organ, a group of organs, anorganelle, a biological signaling pathway (e.g., a receptor-activatedsignaling pathway, a charge-activated signaling pathway, a metabolicpathway, a cellular signaling pathway, etc.), a group of proteins, agroup of nucleic acids, or a group of molecules (including, but notlimited to biomolecules) that carry out at least one biological functionor biological task within cellular membranes, cellular compartments,cells, cell cultures, tissues, organs, organ systems, organisms,multicellular organisms, or any biological entities. In someembodiments, biological systems are cell signaling pathways comprisingintracellular and/or extracellular signaling biomolecules. In someembodiments, biological systems comprise proteolytic cascades (e.g., thecomplement cascade).

Control system: As used herein, the term “control system” refers to abiological system that is untreated and used for comparison to abiological system that is or has been treated or otherwise manipulated.

Downstream event: As used herein, the term “downstream” or “downstreamevent,” refers to any event occurring after and as a result of anotherevent. In some cases, downstream events are events occurring after andas a result of C5 cleavage and/or complement activation. Such events mayinclude, but are not limited to generation of C5 cleavage products,activation of MAC, hemolysis, and hemolysis-related disease (e.g., PNH).

Sample: As used herein, the term “sample” refers to an aliquot orportion taken from a source and/or provided for analysis or processing.In some embodiments, a sample is from a biological source such as atissue, cell or component part (e.g. a body fluid, including but notlimited to blood, mucus, lymphatic fluid, synovial fluid, cerebrospinalfluid, saliva, amniotic fluid, amniotic cord blood, urine, vaginal fluidand semen). In some embodiments, a sample may be or comprise ahomogenate, lysate or extract prepared from a whole organism or a subsetof its tissues, cells or component parts, or a fraction or portionthereof, including but not limited to, for example, plasma, serum,spinal fluid, lymph fluid, the external sections of the skin,respiratory, intestinal, and genitourinary tracts, tears, saliva, milk,blood cells, tumors, organs. In some embodiments, a sample is orcomprises a medium, such as a nutrient broth or gel, which may containcellular components, such as proteins or nucleic acid molecule. In someembodiments, a “primary” sample is an aliquot of the source. In someembodiments, a primary sample is subjected to one or more processing(e.g., separation, purification, etc.) steps to prepare a sample foranalysis or other use.

Subject: As used herein, the term “subject” refers to any organism towhich a compound in accordance with the invention may be administered,e.g., for experimental, diagnostic, prophylactic, and/or therapeuticpurposes. Typical subjects include animals (e.g., mammals such as mice,rats, rabbits, porcine subjects, non-human primates, and humans.)

I. Compounds and Compositions

In some embodiments, the present invention provides compounds andcompositions for modulation of complement activity. In some cases,compounds include C5 modulatory compounds. Such compounds may include,but are not limited to polypeptides (e.g. cyclic polypeptides,peptidomimetics and cyclic peptidomimetics). As used herein, a “mimetic”refers to a molecule which exhibits some of the properties or featuresof another molecule. A “peptidomimetic” or “polypeptide mimetic” is amimetic in which the molecule contains structural elements that are notfound in natural polypeptides (i.e., polypeptides comprised of only the20 proteinogenic amino acids). In some embodiments, peptidomimetics arecapable of recapitulating or mimicking the biological action(s) of anatural peptide. A peptidomimetic may differ in many ways from naturalpolypeptides, including, but not limited to changes in backbonestructure and the presence of amino acids that do not occur in nature.In some cases, peptidomimetics may include amino acids with side chainsthat are not found among the known 20 proteinogenic amino acids,non-polypeptide-based bridging moieties used to effect cyclizationbetween the ends or internal portions of the molecule, substitutions ofthe amide bond hydrogen moiety by methyl groups (N-methylation) or otheralkyl groups, replacement of a peptide bond with a chemical group orbond that is resistant to chemical or enzymatic treatments, N- andC-terminal modifications, and conjugation with a non-peptidic extension(such as polyethylene glycol, lipids, carbohydrates, nucleosides,nucleotides, nucleoside bases, various small molecules, or phosphate orsulfate groups).

Some polypeptides of the invention may be cyclic. Cyclic polypeptidesinclude any polypeptides that have as part of their structure one ormore cyclic features such as a loop, bridging moiety, and/or an internallinkage. As used herein, the term “bridging moiety” refers to one ormore components of a bridge formed between two adjacent or non-adjacentamino acids, unnatural amino acids or non-amino acids in a polypeptide.Bridging moieties may be of any size or composition. In someembodiments, bridging moieties may comprise one or more chemical bondsbetween two adjacent or non-adjacent amino acids, unnatural amino acids,non-amino acid residues or combinations thereof. In some embodiments,such chemical bonds may be between one or more functional groups onadjacent or non-adjacent amino acids, unnatural amino acids, non-aminoacid residues or combinations thereof. Bridging moieties may compriseone or more features including, but not limited to an amide bond(lactam), disulfide bond, thioether bond, aromatic ring, triazole ring,and hydrocarbon chain. In some embodiments, bridging moieties comprisean amide bond between an amine functionality and a carboxylatefunctionality, each present in an amino acid, unnatural amino acid ornon-amino acid residue side chain. In some embodiments, the amine orcarboxylate functionalities are part of a non-amino acid residue orunnatural amino acid residue. In some cases, bridging moieties maycomprise bonds formed between residues that may include, but are notlimited to (S)-2-amino-5-azidopentanoic acid (also referred to herein as“X02”), (S)-2-aminohept-6-enoic acid (also referred to herein as “X30”),(S)-2-aminopent-4-ynoic acid (also referred to herein as “X31”) and(S)-2-aminopent-4-enoic acid (also referred to herein as “X12”.)Bridging moieties may be formed through cyclization reactions usingolefin metathesis. In some cases, such bridging moieties may be formedbetween X12 and X30 residues. In some embodiments, the bridging moietycomprises a disulfide bond formed between two thiol containing residues.In some embodiments, the bridging moiety comprises one or more thioetherbonds. Such thioether bonds, may include those found in cyclo-thioalkylcompounds. These bonds are formed during a chemical cyclization reactionbetween chloro acetic acid (also referred to herein as “X35”) N-terminalmodified groups and cysteine residues. In some cases, bridging moietiescomprise one or more triazole ring. Such triazole rings may include, butare not limited to those formed by cyclization reaction between X02 andX31. In some embodiments, bridging moieties comprise non-protein ornon-polypeptide based moieties, including, but not limited to cyclicrings (including, but not limited to aromatic ring structures (e.g.xylyls)). Such bridging moieties may be introduced by reaction withreagents containing multiple reactive halides, including, but notlimited to poly(bromomethyl)benzenes, poly(bromomethyl)pyridines,poly(bromomethyl)alkylbenzenes and/or (E)-1,4-dibromobut-2-ene. In someembodiments, bridging moieties of the present invention include, but arenot limited to the following structures:

wherein each X is independently N or CH, such that no ring contains morethan 2 N; each Z is independently absent or selected from a bond, NR, O,S, CH₂, C(O)NR, NRC(O), S(O)_(v)NR and NRS(O)_(v); each m isindependently selected from 0, 1, 2, and 3; each v is independentlyselected from 1 and 2; each R is independently selected from H andC₁-C₆; and each bridging moiety is connected to the polypeptide by anindependently selected bond or C₁-C₆ spacer.

In certain embodiments of this invention, polypeptides are renderedmacrocyclic by formation of covalent bonds between atoms present withinthe linear polypeptide and atoms of a bridging moiety. This bridgingmoiety serves the purpose of chemically tethering two reactive sites onthe linear polypeptide so as to furnish a cyclic polypeptide product.Embodiments of the present invention include polypeptides cyclized inthe aforementioned manner and comprising a bridging moiety containing anaromatic, 6-membered ring. In these embodiments, atoms of the linearpolypeptide that form explicit chemical bonds with the bridging moietymay be heteroatoms (including, but not limited to, nitrogen, oxygen andsulfur), or saturated or unsaturated carbon atoms. In each of theseembodiments of this invention, the atoms of the polypeptide side chainmay be bonded directly to a carbon atom within the aromatic ring of thebridging moiety. In alternative forms, the atoms of the polypeptide sidechain may be bonded to a saturated —CH2- group that is in turn directlybonded to a carbon atom within the aromatic ring of the bridging moiety.In certain cases, the aromatic, 6-membered ring within the bridgingmoiety is benzene, as in the following structures wherein Z may beselected from NH, S, O and (CH)₂:

In alternative forms of this invention, the aromatic, 6-membered ringthat comprises the bridging moiety is heterocyclic and contains one ormore nitrogen atoms. In these embodiments, the aromatic heterocycle maybe pyridine, containing a single nitrogen atom in the aromatic ring[e.g. any of the structures below wherein Z may be selected from NH, S,O and (CH)₂]:

Aromatic heterocycles may alternatively be pyridazine, containing twoadjacent nitrogen atoms in a 1,2-orientation within the aromatic ring[e.g. any of the structures below wherein Z may be selected from NH, S,O and (CH)₂]:

In other embodiments, the aromatic heterocycle may be pyrimidine,containing two nitrogen atoms in a 1,3-orientation within the aromaticring [e.g. any of the structures below wherein Z may be selected fromNH, S, O and (CH)₂]:

Alternatively, the aromatic heterocycle may be pyrazine, containing twonitrogen atoms in a 1,4-orientation within the aromatic ring [e.g. anyof the structures below wherein Z may be selected from NH, S, O and(CH)₂]:

In alternative forms of this invention, polypeptides are renderedmacrocyclic as a result of the formation of covalent bonds between atomsof the linear polypeptide and atoms of a bridging moiety consisting of aheterocyclic, aromatic, 5-membered ring. In these embodiments, atoms ofthe linear polypeptide that form explicit chemical bonds with thebridging moiety may be heteroatoms (including, but not limited to,nitrogen, oxygen and sulfur), or saturated or unsaturated carbon atoms.In each of these embodiments of this invention, the atoms of thepolypeptide side chain may be bonded directly to a carbon atom ornitrogen atom within the aromatic ring of the bridging moiety. Inalternative forms, the atoms of the polypeptide side chain may be bondedto a saturated —CH2- group that is in turn directly bonded to a carbonatom or nitrogen atom within the aromatic ring of the bridging moiety.In certain cases, the heterocyclic, aromatic, 5-membered ring within thebridging moiety is 1,2,3-triazole. In these embodiments, the aromaticring may be substituted at positions 1 and 4 with chemical functionalityof the linear polypeptide that are being tethered. Alternatively, the1,2,3-triazole scaffold may be substituted at positions 1 and 4 with—CH2- groups that are directly bonded to the atoms of the linearpolypeptide being tethered [e.g. either of the structures below whereinZ may be selected from NH, S, O and (CH)₂]:

In other embodiments of this invention, the heterocyclic, aromatic,5-membered ring that comprises the bridging moiety is pyrazole. In theseembodiments, the aromatic ring may be substituted either at positions 1and 3 or at positions 1 and 4 with chemical functionality of the linearpolypeptide that are being tethered. Alternatively, the pyrazolescaffold may be substituted either at positions 1 and 3 or at positions1 and 4 with —CH₂— groups that are directly bonded to the atoms of thelinear polypeptide being tethered [e.g. any of the structures belowwherein Z may be selected from NH, S, O and (CH)₂]:

Unless otherwise defined, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention belongs. Although methods and materialssimilar or equivalent to those described herein can be used in thepractice or testing of the cyclic polypeptides and methods featured inthe invention, suitable methods and materials are described below.

Polypeptides as Drugs

By virtue of their size and complexity, polypeptides are able to formnumerous, highly specific contacts with their biological targets and canshow a high level of selectivity for the correct or desired target ascompared to a closely related target within the same family. Off-targeteffects (known also as side effects) often cause highly effective drugsto fail regulatory approval due to safety concerns.

Numerous polypeptides (including, but not limited to peptidomimetics,have been developed into effective drugs. These include, but are notlimited to, insulin, glucagon-like peptide 1 (GLP-1), somatostatin,vasopressin, cyclosporine A, and the like. The therapeutic polypeptidecan be identical to the naturally occurring molecule (i.e. that whichcirculates in humans and is considered “wild-type” in the humanpopulation). In many other cases, the polypeptide is not suitable orsub-optimal for therapeutic use due to a short circulating half-lifethat is often due to metabolic instability in the body. In these cases amodified or a variant form of the polypeptide (peptidomimetic) is usedwhich results in improved pharmacokinetic and pharmacodynamic behavior.In other cases a polypeptide derived from a natural source has anequivalent mechanism of action and a preferred pharmaceutical profileand can be used as a therapy. For example, exenatide, a syntheticversion of exedin-4, has biological properties similar to humanglucagon-like peptide-1 (GLP-1) but improved pharmacokinetics, and hasbeen approved by the FDA for the treatment of diabetes mellitus type 2.As another example, salmon calcitonin, calcitonin extracted from theUltimobranchial glands of salmon, resembles human calcitonin but is moreactive than human calcitonin and may be used to treat postmenopausalosteoporosis, hypercalcaemia, Paget's disease, bone metastases andphantom limb pain.

Polypeptides are typically limited to non-oral routes of administration.In nearly all cases, polypeptides must be delivered by injection, sinceeven very short polypeptides (e.g., polypeptides with 4-10 amino acidresidues) are incapable or poorly capable of passing through the cellmembranes lining the intestinal tract. For efficient oral availability,drugs typically need to pass through both the luminal and basolateralmembranes of gut epithelial cells in order to enter the systemiccirculation. The poor membrane permeability and lack of oralbioavailability of polypeptides significantly limits their therapeuticuse.

The effectiveness of a polypeptide as a drug may be influenced by itsproteolytic stability. Within the body, polypeptides can be modified ordegraded by enzymes, which can limit their effectiveness for interactingwith an intended target.

Metabolic stability of polypeptides is important as it is related totheir global flexibility, intramolecular fluctuations, various internaldynamic processes as well as many biologic functions. The metabolicstability of polypeptides may be critical in the development ofpharmaceuticals, affecting parameters such as, but not limited to,clearance, half-life and bioavailability of the drugs.

Maintaining a given level of a therapeutic polypeptide within the bodyor the bloodstream may be difficult due to efflux. The rate of efflux ofa polypeptide from the body may vary and should be monitored whenconsidering the administration of therapeutic polypeptides.

There remains a significant medical need for complement activationinhibitors or inhibitors of complement activity and inhibitorformulations that are highly potent and highly specific.

Discovery of Peptidomimetics

Peptidomimetics may be identified by a variety of means. In some cases anaturally occurring peptide or a sequence found in a natural protein isused as a starting point. In these instances, the starting peptidesequence has been chosen because it is known to physically interact witha desired target molecule. A natural peptide may be chosen because it isan agonist or antagonist for a receptor, inhibits an enzyme, ormodulates a channel. A sequence found in a natural protein may be chosenbecause it comprises a domain that participates in an interaction withanother protein or some other molecule in a human or animal. In manycases, structural data on interacting proteins can be obtained frompublic databases (e.g. the RCSB Protein Data Bank; H. M. Berman, J.Westbrook, Z. Feng, G. Gilliland, T. N. Bhat, H. Weissig, I. N.Shindyalov, P. E. Bourne (2000) The Protein Data Bank Nucleic AcidsResearch, 28: 235-242) and the specific region of a protein thatinteracts with the desired target can be identified fromcrystallographic data on the protein complex. In other cases,polypeptides corresponding to various portions of a protein can beprepared and tested for binding to a target of interest. Onceidentified, chemical modifications are introduced to improve itsstability and potency, with the resulting peptidomimetic having improvedpharmacokinetic or pharmacodynamic parameters.

In other cases, a polypeptide is isolated by one of several methods forisolating polypeptide sequences from libraries of polypeptides based ontheir affinities to specific target proteins, nucleic acids,carbohydrates, lipids, or whole cells. Such methods include phagedisplay, mRNA display, ribosome display, DNA display, DNA-encodedassembly, and two-hybrid screening, as well as their modifications (See,e.g., Takashashi, T. T et al. (2003). Trends in Biochem. Sci.28(3):159-165; Kay, B. K. et al. (2001). Methods. 24:240-246; He, M andTaussig, M (2002). Briefs in Functional Genomics and Proteomics. 1(2):204-212; Rothe, A. et al. (2006). The FASEB Journal. 20(10):1599-1610;all of which are included herein by reference in their entireties.)

Polypeptides can adopt three-dimensional structures that are capable ofbinding to other biological molecules with certain degrees of affinityand specificity. Some will bind with very high affinity and specificity.A library of random polypeptide sequences will be populated by moleculeswith a wide variety of three-dimensional structures. In order to isolatea polypeptide with a conformation that interacts with a specific targetprotein, individual sequences from the library can be prepared andtested or screened for their affinity to the target. However, for verylarge libraries (>10⁶ members), the screening of individual sequencesfor binding affinity is not feasible. To overcome this limitation, anumber of techniques have been developed to select novel polypeptidesfrom extremely large, complex mixtures by virtue of their bindingaffinity to a target. Since high affinity binding polypeptides arepredicted to be present at a very low frequency within the population,these selection methods rely on maintaining a physical link between thepolypeptide and the genetic material (generally a nucleic acid such asDNA or RNA) encoding the polypeptide so that selection of thepolypeptide automatically includes selection of a nucleic acid encodingit. The nucleic acid encoding the selected polypeptide can be amplifiedand sequenced to reveal the sequence of both the nucleic acid and thepolypeptide. In one approach, phage display (see Cwirla, S. E. et al.(1990). Proc. Natl. Acad. Sci. U.S.A. 87:6378-6382; Dower, W. J. andCwirla, S. E. U.S. Pat. Nos. 5,427,908 and 5,580,717), each randompolypeptide member of the library is displayed on the surface of abacteriophage particle as part of a fusion protein between thepolypeptide and one of the phage coat proteins. The phage particleprovides the link between the polypeptide and the encoding DNA byco-localizing them within the same physical entity, and the encoding DNAcan be subsequently amplified by infecting bacteria with the selectedphage. In another approach, ribosome display (see Kawasaki, G. H. U.S.Pat. Nos. 5,658,754 and 5,643,768), a mixture of messenger RNA (mRNA)molecules is translated in vitro in a manner that produces, for eachmRNA in the mixture, a stabilized complex of ribosome, mRNA, and newlysynthesized polypeptide protruding from the ribosome. Stabilizing thecomplex permits it to be held together while the polypeptides arescreened for binding to a target of interest. The mRNAs encoding theselected polypeptides can be amplified using polymerase chain reaction(PCR), and then characterized, e.g., by sequencing.

In yet another approach, mRNA display (see Szostak, J. W. and Roberts,R. W., U.S. Pat. No. 6,258,558, the contents of which are incorporatedherein by reference in their entirety), each mRNA molecule in thelibrary is modified by the covalent addition of a puromycin-like moietyat its 3′ terminus. The puromycin-like moiety is an aminoacyl-tRNAacceptor stem analog that functions as a peptidyl acceptor, and can beadded to a growing polypeptide chain by the peptidyl transferaseactivity of a ribosome translating the mRNA. During in vitrotranslation, the mRNA and the encoded polypeptide become covalentlylinked through the puromycin-like moiety, creating an RNA-peptidefusion. After selecting a fusion molecule by binding of its polypeptidecomponent to a target, the RNA component of the selected fusion moleculecan be amplified using PCR, and then characterized. Several othermethods have been developed to produce a physical linkage between apolypeptide and its encoding nucleic acid to facilitate selection andamplification (see Yanagawa, H., Nemoto, N., Miyamoto, E., and Husimi,Y., U.S. Pat. No. 6,361,943; Nemoto, H., Miyamoto-Sato, E., Husimi, H.,and Yanagawa, H. (1997). FEBS Lett. 414:405-408; Gold, L., Tuerk, C.,Pribnow, D., and Smith, J. D., U.S. Pat. Nos. 5,843,701 and 6,194,550;Williams, R. B., U.S. Pat. No. 6,962,781; Baskerville, S. and Bartel, D.P. (2002). Proc. Natl. Acad. Sci. USA 99:9154-9159; Baskerville, D. S.and Bartel, D. P., U.S. Pat. No. 6,716,973; Sergeeva, A. et al. (2006).Adv. Drug Deliv. Rev. 58:1622-1654; the contents of each of which areincorporated herein by reference in their entirety).

mRNA display is a particularly useful method for creating largelibraries of polypeptides. Accordingly, provided herein are methods ofselecting for a polypeptide (or an mRNA encoding a polypeptide) thatinteracts with complement protein C5. A library will generally containat least 10² members, more preferably at least 10⁶ members, and morepreferably at least 10⁹ members (e.g., any of the mRNA-polypeptidecomplexes). In some embodiments, the library will include at least 10¹²members or at least 10¹⁴ members. In general, the members will differfrom each other; however, it is expected there will be some degree ofredundancy in any library. The library can exist as a single mixture ofall members, or can be divided into several pools held in separatecontainers or wells, each containing a subset of the library, or thelibrary can be a collection of containers or wells on a plate, eachcontainer or well containing just one or a few members of the library.

Each mRNA in the library preferably comprises a translation initiationsequence, a start codon, and a variable polypeptide (e.g., protein orshort peptide) coding region that is generated by, for example, a randomor semi-random assembly of nucleotides, and varies from mRNA to mRNA inthe library (though there will likely be some degree of redundancywithin the library). The translation initiation sequence, start codon,and variable polypeptide coding region can be flanked by known, fixedsequences that can be used for PCR amplification of the mRNA, e.g.,after selection. Other fixed sequences that can be present include thosecorresponding to sequences that encode amino acids that can participatein chemical or enzymatic cross-linking reactions, such that thepolypeptide produced can be modified or derivatized after translation,or that encode a fixed C-terminal extension such as a polypeptide tagthat can facilitate purification of the peptide-mRNA fusions.

Once a library of mRNA derivatized with puromycin is generated, thelibrary can be translated. The resulting polypeptides (e.g., displayedpolypeptides) will be linked to their corresponding mRNAs as describedherein (e.g., as an mRNA-polypeptide complex).

Numerous in vitro translation systems have been described in theliterature. The most common systems utilize rabbit reticulocyte lysates,wheat germ extracts, or E. coli extracts, which are available from anumber of commercial sources in kit form (e.g., Ambion, Austin, Tex.;Promega, Madison, Wis.; Novagen/EMD Chemicals, Gibbstown, N.J.; Qiagen,Valencia, Calif.).

Unlike phage display or other systems that rely on translation withincells, mRNA display can be adapted to directly produce libraries ofpeptidomimetics by performing in vitro translation with unnatural ornon-standard amino acids. The 20 natural proteinogenic amino acids areidentified and referred to herein by either the one-letter orthree-letter designations as follows: aspartic acid (Asp:D), isoleucine(Ile:I), threonine (Thr:T), leucine (Leu:L), serine (Ser:S), tyrosine(Tyr:Y), glutamic acid (Glu:E), phenylalanine (Phe:F), proline (Pro:P),histidine (His:H), glycine (Gly:G), lysine (Lys:K), alanine (Ala:A),arginine (Arg:R), cysteine (Cys:C), tryptophan (Trp:W), valine (Val:V),glutamine (Gln:Q) methionine (Met:M), asparagine (Asn:N). Naturallyoccurring amino acids exist in their levorotary (L) stereoisomericforms. Amino acids referred to herein are L-stereoisomers except whereotherwise indicated

Unnatural amino acids have side chains or other features not present inthe 20 naturally-occurring amino acids listed above and include, but arenot limited to: N-methyl amino acids, N-alkyl amino acids, alpha, alphasubstituted amino acids, beta-amino acids, alpha-hydroxy amino acids,D-amino acids, and other unnatural amino acids known in the art (See,e.g., Josephson et al., (2005) J. Am. Chem. Soc. 127: 11727-11735;Forster, A. C. et al. (2003) Proc. Natl. Acad. Sci. USA 100: 6353-6357;Subtelny et al., (2008) J. Am. Chem. Soc. 130: 6131-6136; Hartman, M. C.T. et al. (2007) PLoS ONE 2:e972; and Hartman et al., (2006) Proc. Natl.Acad. Sci. USA 103:4356-4361).

Essentially any amino acid that, when attached to an appropriate tRNA,can be assembled into a polymer by natural or mutant ribosomes can beused (see Sando, S. et al., (2007) J. Am. Chem. Soc. 129:6180-6186;Dedkova, L. et al. (2003) J. Am. Chem. Soc. 125: 6616-6617; Josephson,K., Hartman, M. C. T., and Szostak, J. W. (2005) J. Am. Chem. Soc.127:11727-11735; Forster, A. C. et al. (2003) Proc. Natl. Acad. Sci. USA100:6353-6357; Subtelny, A. O., Hartman, M. C. T., and Szostak, J. W.(2008) J. Am. Chem. Soc. 130:6131-6136; and Hartman, M. C. T. et al.(2007) PLoS ONE 2:e972).

When unnatural amino acids are desired, it may be advantageous to use apurified translation system that lacks endogenous aminoacylated tRNAs(Shimizu, Y. et al. (2001) Nat. Biotech. 19:751-755; Josephson, K.,Hartman, M. C. T., and Szostak, J. W. (2005) J. Am. Chem. Soc. 127:11727-11735; Forster, A. C. et al. (2003) Proc. Natl. Acad. Sci. USA100: 6353-6357). If unnatural amino acids are used with an in vitrotranslation system based on a lysate or extract, it may be desirable todeplete the extract of endogenous tRNAs, as previously described (seeJackson, R. J., Napthine, S., and Brierley, I. (2001) RNA 7:765-773). Asystem based on purified E. coli translation factors is commerciallyavailable (PUREXPRESS™; New England Biolabs, Ipswich, Mass.). Thesesystems are particularly useful for translation with unnatural aminoacids to produce peptidomimetics.

When using natural amino acids with an in vitro translation system basedon a lysate or extract, translation is dependent on the enzymaticcharging of amino acids onto tRNAs by tRNA synthetases, all of which arecomponents of the extracts. Alternatively, in vitro translation systemsthat use purified translation factors and ribosomes, or tRNA-depletedextracts, require that aminoacylated tRNAs be provided. In theseinstances, purified or in vitro synthesized tRNAs can be charged withamino acids using chemical (see Frankel, A., Millward, S. W., andRoberts, R. W. (2003) Chem. Biol. 10:1043-1050) or enzymatic procedures(Josephson, K., Hartman, M. C. T., and Szostak, J. W. (2005) J. Am.Chem. Soc. 127: 11727-11735; Murakami, H. et al. (2006) Nat. Methods3:357-359).

Numerous publications describe the recovery of mRNA-displayedpolypeptides from translation complexes, and these are suitable for usewith the methods described herein (Liu, R. et al. (2000). MethodsEnzymol. 318:268-293; Baggio, R. et al. (2002). J. Mol. Recognit.15:126-134; U.S. Pat. No. 6,261,804). The recovery of mRNA-displayedpolypeptides may be facilitated by the use of various “tags” that areincluded in the polypeptide by translation of fixed sequences of thepolypeptide coding sequence and which bind to specific substrates ormolecules. Numerous reagents for capturing such tags are commerciallyavailable, including reagents for capturing the His-tag, FLAG-tag,glutathione-S-transferase (GST) tag, strep-tag, HSV-tag, T7-tag, S-tag,DsbA-tag, DsbC-tag, Nus-tag, myc-tag, hemagglutinin (HA)-tag, or Trx-tag(Novagen, Gibbstown, N.J.; Pierce, Rockford, Ill.). mRNA-displayedpolypeptides can also be isolated by binding of a polyA tail on the mRNAto polydT resin, or a combination of a polyA tail and a His-tag.

After the in vitro translation reaction has been performed, and prior tothe selection step, the mRNA portion of the functionalized RNA istypically reversed-transcribed to produce a RNA-DNA hybrid molecule.This serves to protect the RNA from degradation, and also prevents theRNA from folding into a secondary structure that could bind to theselection target, which would lead to selection of inappropriateproducts (e.g., the selection of RNA aptamers rather than polypeptideaptamers).

After in vitro translation and isolation of polypeptide-mRNA fusions,the polypeptide moiety may be modified by intramolecular orintermolecular cross-linking, chemical conjugation, enzymatic cleavage,truncation, or extension with additional amino acid monomers. One way toaccomplish this is by incorporating unnatural amino acids with reactiveside chains into the polypeptides that make up the library. Aftertranslation, the newly formed polypeptides can be reacted with moleculesthat react specifically with the reactive side chain of the incorporatedamino acid. For example, an amino acid with a terminal alkyne side chaincan be incorporated into the polypeptide library and subsequentlyreacted with an azido sugar, creating a library of displayedpolypeptides with sugars attached at the positions of the alkynyl sidechains (Josephson, K., Hartman, M. C. T., and Szostak, J. W. (2005) J.Am. Chem. Soc. 127: 11727-11735). A variety of reactive side chains canbe used for such post-translational conjugation, including amines,carboxyl groups, azides, terminal alkynes, alkenes, and thiols.

One particularly useful modification is based on the cross-linking ofamino acids to produce cyclic structures. Cyclic regions in apolypeptide contain a rigid domain, which reduces conformationalflexibility and degrees of rotational freedom, leading to very highaffinity binding to target proteins. A number of methods for cyclizing apolypeptide are available to those skilled in the art and areincorporated herein by reference. Typically, the chemical reactivity ofspecific amino acid side chains and/or the carboxyl or amino termini ofthe polypeptide are exploited to crosslink two sites of the polypeptideto produce a cyclic molecule. In one method, the thiol group of acysteine residue is cross-linked with another cysteine residue to form adisulfide bond. In some embodiments, thiol groups of cysteine residuesreact with bromomethyl groups of poly(bromomethyl)benzene molecules toform stable linkages (see Timmerman, P. et al., (2005) ChemBioChem6:821-824, the contents of which are herein incorporated by reference intheir entirety). Poly(bromomethyl)benzene molecules of the presentinvention may include, but are not limited to1,2-bis(bromomethyl)benzene, 1,3-bis(bromomethyl)benzene and1,4-bis(bromomethyl)benzene. Bis-, tris- andtetrakis(bromomethyl)benzene molecules, for example, can be used togenerate bridging moieties to produce polypeptides with one, two orthree loops, respectively. Bromomethyl groups of apoly(bromomethyl)benzene molecule may be arranged on the benzene ring onadjacent ring carbons (ortho- or o-), with a ring carbon separating thetwo groups (meta- or m-) or on opposite ring carbons (para- or p-). Insome embodiments, m-bis(bromomethyl)benzene (also referred to herein asm-dibromoxylene) is utilized in the formation of cyclic polypeptides. Insome embodiments, o-bis(bromomethyl)benzene (also referred to herein aso-dibromoxylene) or p-bis(bromomethyl)benzene (also referred to hereinas p-dibromoxylene) are utilized in the formation of cyclicpolypeptides. In some embodiments, thiol groups of cysteine residuesreact with other reagents comprising one or more bromo functional groupsto form stable linkages. Such reagents may include, but are not limitedto poly(bromomethyl)pyridines (including, but not limited to2,6-bis(bromomethyl)pyridine), poly(bromomethyl)alkylbenzenes(including, but not limited to 1,2-bis(bromomethyl)-4-alkylbenzene)and/or (E)-1,4-dibromobut-2-ene.

In another exemplary method, a side chain amino group and a terminalamino group are cross-linked with disuccinimidyl glutarate (seeMillward, S. W. et al., J. Am. Chem. Soc. 127:14142-14143, 2005). Inother approaches, cyclization is accomplished by forming a thioetherbond between two sites on the polypeptide (see Timmerman, P. et al.,(2005) ChemBioChem 6:821-824; incorporated by reference herein in itsentirety). An enzymatic method relies on the reaction between (1) acysteine and (2) a dehydroalanine or dehydrobutyrine group, catalyzed bya lantibiotic synthetase, to create the thioether bond (see Levengood,M. R. and Van der Donk, W. A., Bioorg. and Med. Chem. Lett.18:3025-3028, 2008). The dehydro functional group can also be generatedchemically by the oxidation of selenium containing amino acid sidechains incorporated during translation (see Seebeck, F. P. and Szostak,J. W. J. Am. Chem. Soc. 2006).

A library of mRNA-polypeptide fusions (also referred to herein as anmRNA display library) generated using the methods described above, andwhich may or not have been subjected to a post-translationalmodification (such as cyclization of the polypeptide, as describedabove), can be subjected to a batch selection step to isolate thosecomplexes displaying desirable polypeptides.

Typically, C5 is conjugated to a solid substrate, such as an agarose orsynthetic polymer bead. Numerous methods are available for immobilizingC5 to a solid support. In one particularly useful method, C5 isconjugated to biotin and streptavidin beads are used to immobilize theprotein. The beads comprising the immobilized C5 are mixed with the mRNAdisplay library and incubated under conditions (e.g., temperature, ionicstrength, divalent cations, and competing binding molecules) that permitspecific members of the library to bind the target. Alternatively, thebiotinylated enzyme can be free in solution and, after binding to anappropriate polypeptide, the mRNA-polypeptide fusions bound to C5 arecaptured by appropriately modified beads.

The binding conditions can be varied in order to change the stringencyof the selection. For example, low concentrations of a competitivebinding agent can be added to ensure that the selected polypeptides havea relatively higher affinity. Alternatively, the incubation period canbe chosen to be very brief, such that only polypeptides with high k_(on)rates (rate of association) will be isolated. In this manner, theincubation conditions play an important role in determining theproperties of the selected polypeptides. Negative selections can also beemployed. In this case, a selection to remove polypeptides with affinityto the substrate to which the target is bound (e.g., Sepharose) iscarried out by applying the displayed library to substrate beads lackingthe target protein. This step can remove mRNAs and their encodedpolypeptides that are not specific for the target protein. Numerousreferences describing how to conduct selection experiments areavailable. (See, e.g., U.S. Pat. No. 6,258,558, Smith, G. P. andPetrenko, V. A., (1997) Chem. Rev. 97:391-410; Keefe, A. D. and Szostak,J. W. (2001) Nature 15:715-718; Baggio, R. et al. (2002) J. Mol. Recog.15:126-134 and Sergeeva, A. et al. (2006) Adv. Drug Deliv. Rev.58:1622-1654; the contents of each of which are herein incorporated byreference in their entirety).

The frequency at which binding molecules are present in a library ofrandom sequences is expected to be very low. Thus, in the initialselection step, very few polypeptides meeting the selection criteria(and their associated mRNAs) should be recovered. Typically, theselection is repeated with mRNAs selected from the first round ofselection. This is accomplished by using PCR to amplify the mRNAs orcorresponding cDNAs selected in the first round, followed by in vitrotranscription to produce a new library of mRNAs. PCR primerscorresponding to the 5′ and 3′ ends of the mRNAs in the library areused. Typically, the 5′ primer will extend in the 5′ direction beyondthe end of the mRNA so that a bacterial promoter, such as a T7 promoter,is added to the 5′ end of each amplified molecule. Once amplified, thedouble-stranded DNA can be used in an in vitro transcription reaction togenerate the mRNA for a subsequent round of selection.

The selection process typically involves a number of rounds or cycles,in which the pool of selected molecules is incrementally enriched in aspecific set of sequences at the end of each round. The selectionconditions may be the same for each round, or the conditions may change,for example, in order to increase the stringency of selection in laterrounds. The progress of selection may be monitored by the use ofisotopically-labeled amino acids, such as ³⁵S methionine. The amount ofradiolabeled polypeptide bound to the target at each round is measured,and a progressive increase in recovered radiolabel is indicative of aprogressive enrichment in RNA molecules encoding polypeptides withbinding affinity to the target. After any round, the PCR products may becloned and sequenced. Generally, cloning and sequencing is performedafter a round in which appreciable (e.g. >2% over background to beadslacking immobilized C5) amounts of radiolabeled polypeptide arerecovered in the target-bound pool. Sequences that are found in multipleisolates are candidates for encoding polypeptides that bind specificallyto the target. Alternatively, high throughput sequencing of thousands ofclones can be performed after the first or subsequent rounds. Sequencesthat increase in frequency between, for example, the third and fourthrounds are candidates for encoding polypeptides that bind specificallyto the target. The polypeptide encoded by any sequence may be translatedor synthesized and tested for binding affinity to the original targetprotein used in the selection.

The libraries and methods of the present invention may be used tooptimize the function or properties of a polypeptide. In one approach,mutagenic PCR (Keefe, A. D. and Szostak, J. W. (2001). Nature15:715-718) is used to introduce sequence variation in the library oncethe population is enriched in polypeptides with a certain level ofbinding affinity. Alternatively, a single RNA sequence encoding apolypeptide with defined binding properties can be replicated but with adefined level of mutations, or mutagenic PCR can be performed to producea pool of mutant molecules. Upon in vitro translation the resultingmixture of mRNA molecules produced from such a pool is expected toencode polypeptides with a range of improved, similar, or reducedaffinities as compared to the starting sequence, and a selectionperformed on mRNAs from such a pool may be expected to identifypolypeptides with improved affinity if an appropriate stringency regimenis used during the selection.

In a second approach, optimization is performed in a directed manner. Asequence encoding a polypeptide with established binding or functionalproperties is subjected to site-directed mutagenesis, whereby a seriesof sequences is produced, with each sequence having one codon replacedwith, for example, an alanine codon. The number of sequences in the setis equal to the number of amino acid residues that are to be mutated.After in vitro translation, the polypeptide product of each “alaninescanning” mutant is tested for binding or functional properties. Sitesat which an alanine substitution affects the binding or function of thepolypeptide are considered critical residues. Similarly, an N-methylscan may be performed, such that each residue is replaced with theN-methyl derivative, and positions in the polypeptide backbone that cantolerate N-methyl substitutions can be identified.

Alternatively, the sequences can be pooled, subjected to one or morerounds of a high stringency selection, and a pool of sequencesrepresenting high affinity binding polypeptides is isolated. Criticalresidues are identified after DNA sequencing of the recovered DNA asthose that cannot be substituted by an alanine residue without loss ofactivity. Once the critical residues are identified, a pool of mRNAmolecules encoding a wide variety of natural (or unnatural) amino acidsat each critical position is produced. The resulting pool is subjectedto one or more rounds of a high stringency selection (with theappropriate mixture of tRNAs charged with natural or unnatural aminoacids), and sequences representing high affinity binding polypeptidesare isolated after in vitro translation. In this manner, an optimalpolypeptide can be identified. Since the optimal sequence may notnecessarily be identified by combining optimal residues at individualsites, it is useful to test mutations at multiple sites in combination.

Both alanine and N-methyl scanning can also be performed using chemicalsynthesis approaches, such as solid phase polypeptide synthesis (seee.g., Coin, I et al. (2007); Nature Protocols 2(12):3247-56, thecontents of which are herein incorporated by reference in theirentirety).

Once a pool, population or subset of polypeptides is identified, theymay be evaluated for therapeutic or diagnostic applications, includingimproved pharmacokinetic and/or pharmacodynamic properties.

In one embodiment, polypeptides are evaluated for one or more of targetbinding affinity, activity in biochemical or cell based assays, proteaseresistance, in vitro or in vivo permeability, properties related tosuitability for use as a pharmaceutical agent such as plasma proteinbinding, metabolism (in microsomes, hepatocytes, or plasma),P-glycoprotein (Pgp) inhibition and Cytochrome P450 inhibition.Polypeptides of the present invention may also undergo testing for oralbioavailability, toxicity, human ether-a-go-go related gene product(hERG) inhibition, circulating half-life, other pharmacokinetic andpharmacodynamic parameters, and efficacy in animal models of disease.

Polypeptides of the Invention

According to the present invention, once a single polypeptide or a poolof candidate polypeptide molecules is identified, they may undergo oneor more rounds of structure-activity relationship (SAR) optimizationusing standard chemical and polypeptide synthesis techniques. Suchoptimization may include considerations such as avoiding charged polarside chains (Asp, Glu, Arg, Lys) that may inhibit cell penetration,avoidance of side chains that pose metabolic liabilities (Tyr, Met, Trp,Cys), improving solubility, avoidance of unnecessary molecular weight,avoidance of rotatable bonds, and altering lipophilicity.

In one embodiment, it is a goal of the present invention to providecyclic peptidomimetics designed to be metabolically stable, cellpermeable, and/or orally available.

According to some embodiments, polypeptides of the invention comprisefrom about 10 to about 18 amino acids or amino acid variants. In somecases, such polypeptides comprise a cyclic loop.

Amino Acid Variants

As used herein, the term “amino acid” includes the residues of thenatural amino acids as well as unnatural amino acids. The term alsoincludes amino acids bearing a conventional amino protecting group (e.g.acetyl or benzyloxycarbonyl), as well as natural and unnatural aminoacids protected at the carboxy terminus (e.g., as a (C1-C6) alkyl,phenyl or benzyl ester or amide; or as an alpha-methylbenzyl amide).Other suitable amino and carboxy protecting groups are known to thoseskilled in the art (See for example, Greene, T. W.; Wutz, P. G. M.,Protecting Groups In Organic Synthesis; second edition, 1991, New York,John Wiley & sons, Inc., and documents cited therein). Polypeptidesand/or polypeptide compositions of the present invention may alsoinclude modified amino acids.

Unnatural amino acids useful for the optimization of polypeptides and/orpolypeptide compositions of the present invention include, but are notlimited to 1,2,3,4-tetrahydroisoquinoline-1-carboxylic acid,1-amino-2,3-hydro-1H-indene-1-carboxylic acid, homolysine, homoarginine,homoserine, 2-aminoadipic acid, 3-aminoadipic acid, beta-alanine,aminopropionic acid, 2-aminobutyric acid, 4-aminobutyric acid,5-aminopentanoic acid, 5-aminohexanoic acid, 6-aminocaproic acid,2-aminoheptanoic acid, 2-aminoisobutyric acid, 3-aminoisobutyric acid,2-aminopimelic acid, desmosine, 2,3-diaminopropionic acid,N-ethylglycine, N-ethylasparagine, homoproline, hydroxylysine,allo-hydroxylysine, 3-hydroxyproline, 4-hydroxyproline, isodesmosine,allo-isoleucine, N-methylpentylglycine, naphthylalanine, ornithine,pentylglycine, thioproline, norvaline, tert-butylglycine, phenylglycine,azatryptophan, 5-azatryptophan, 7-azatryptophan, 4-fluorophenylalanine,penicillamine, sarcosine, homocysteine, 1-aminocyclopropanecarboxylicacid, 1-aminocyclobutanecarboxylic acid, 1-aminocyclopentanecarboxylicacid, 1-aminocyclohexanecarboxylic acid,4-aminotetrahydro-2H-pyran-4-carboxylic acid,(S)-2-amino-3-(1H-tetrazol-5-yl)propanoic acid, cyclopentylglycine,cyclohexylglycine, cyclopropylglycine, q-co-methyl-arginine,4-chlorophenylalanine, 3-chlorotyrosine, 3-fluorotyrosine,5-fluorotryptophan, 5-chlorotryptophan, citrulline,4-chloro-homophenylalanine, homophenylalanine,4-aminomethyl-phenylalanine, 3-aminomethyl-phenylalanine, octylglycine,norleucine, tranexamic acid, 2-amino pentanoic acid, 2-amino hexanoicacid, 2-amino heptanoic acid, 2-amino octanoic acid, 2-amino nonanoicacid, 2-amino decanoic acid, 2-amino undecanoic acid, 2-amino dodecanoicacid, aminovaleric acid, and 2-(2-aminoethoxy)acetic acid, pipecolicacid, 2-carboxy azetidine, hexafluoroleucine, 3-Fluorovaline,2-amino-4,4-difluoro-3-methylbutanoic acid, 3-fluoro-isoleucine,4-fluoroisoleucine, 5-fluoroisoleucine, 4-methyl-phenylglycine,4-ethyl-phenylglycine, 4-isopropyl-phenylglycine,(S)-2-amino-5-azidopentanoic acid (also referred to herein as “X02”),(S)-2-aminohept-6-enoic acid (also referred to herein as “X30”),(S)-2-aminopent-4-ynoic acid (also referred to herein as “X31”),(S)-2-aminopent-4-enoic acid (also referred to herein as “X12”),(S)-2-amino-5-(3-methylguanidino) pentanoic acid,(S)-2-amino-3-(4-(aminomethyl)phenyl)propanoic acid,(S)-2-amino-3-(3-(aminomethyl)phenyl)propanoic acid,(S)-2-amino-4-(2-aminobenzo[d]oxazol-5-yl)butanoic acid, (S)-leucinol,(S)-valinol, (S)-tert-leucinol, (R)-3-methylbutan-2-amine,(S)-2-methyl-1-phenylpropan-1-amine, and(S)—N,2-dimethyl-1-(pyridin-2-yl)propan-1-amine,(S)-2-amino-3-(oxazol-2-yl)propanoic acid,(S)-2-amino-3-(oxazol-5-yl)propanoic acid,(S)-2-amino-3-(1,3,4-oxadiazol-2-yl)propanoic acid,(S)-2-amino-3-(1,2,4-oxadiazol-3-yl)propanoic acid,(S)-2-amino-3-(5-fluoro-1H-indazol-3-yl)propanoic acid, and(S)-2-amino-3-(1H-indazol-3-yl)propanoic acid,(S)-2-amino-3-(oxazol-2-yl)butanoic acid, (S)-2-amino-3-(oxazol-5-yl)butanoic acid, (S)-2-amino-3-(1,3,4-oxadiazol-2-yl) butanoic acid,(S)-2-amino-3-(1,2,4-oxadiazol-3-yl) butanoic acid,(S)-2-amino-3-(5-fluoro-1H-indazol-3-yl) butanoic acid, and(S)-2-amino-3-(1H-indazol-3-yl) butanoic acid, 2-(2′MeOphenyl)-2-aminoacetic acid, tetrahydro 3-isoquinolinecarboxylic acid and stereoisomersthereof (including, but not limited, to D and L isomers).

Additional unnatural amino acids that are useful in the optimization ofpolypeptides or polypeptide compositions of the invention include butare not limited to fluorinated amino acids wherein one or more carbonbound hydrogen atoms are replaced by fluorine. The number of fluorineatoms included can range from 1 up to and including all of the hydrogenatoms. Examples of such amino acids include but are not limited to3-fluoroproline, 3,3-difluoroproline, 4-fluoroproline,4,4-difluoroproline, 3,4-difluroproline, 3,3,4,4-tetrafluoroproline,4-fluorotryptophan, 5-flurotryptophan, 6-fluorotryptophan,7-fluorotryptophan, and stereoisomers thereof.

Further unnatural amino acids that are useful in the optimization ofpolypeptides or polypeptide compositions of the invention include butare not limited to those that are disubstituted at the α-carbon. Theseinclude amino acids in which the two substituents on the α-carbon arethe same, for example α-amino isobutyric acid, and 2-amino-2-ethylbutanoic acid, as well as those where the substituents are different,for example α-methylphenylglycine and α-methylproline. Further thesubstituents on the α-carbon may be taken together to form a ring, forexample 1-aminocyclopentanecarboxylic acid, 1-aminocyclobutanecarboxylicacid, 1-aminocyclohexanecarboxylic acid,3-aminotetrahydrofuran-3-carboxylic acid,3-aminotetrahydropyran-3-carboxylic acid,4-aminotetrahydropyran-4-carboxylic acid,3-aminopyrrolidine-3-carboxylic acid, 3-aminopiperidine-3-carboxylicacid, 4-aminopiperidinnne-4-carboxylix acid, and stereoisomers thereof.

Additional unnatural amino acids that are useful in the optimization ofpolypeptides or polypeptide compositions of the invention include butare not limited to analogs of tryptophan in which the indole ring systemis replaced by another 9 or 10 membered bicyclic ring system comprising0, 1, 2, 3 or 4 heteroatoms independently selected from N,O, or S. Eachring system may be saturated, partially unsaturated or fullyunsaturated. The ring system may be substituted by 0, 1, 2, 3, or 4substituents at any substitutable atom. Each substituent isindependently selected from H, F, Cl, Br, CN, COOR, CONRR′, oxo, OR,NRR′. Each R and R′ is independently selected from H, C1-C20 alkyl,C1-C20 alkyl-O—C1-20 alkyl.

In some embodiments, analogs of tryptophan (also referred to herein as“tryptophan analogs”) that are useful in the optimization ofpolypeptides or polypeptide compositions of the invention include, butare not limited to 5-fluorotryptophan [(5-F)W], 5-methyl-O-tryptophan[(5-MeO)W], 1-methyltryptophan [(1-Me-W) or (1-Me)W], D-tryptophan(D-Trp), azatryptophan (including, but not limited to 4-azatryptophan,7-azatryptophan and 5-azatryptophan,) 5-chlorotryptophan,4-fluorotryptophan, 6-fluorotryptophan, 7-fluorotryptophan, andstereoisomers thereof. Except where indicated to the contrary, the term“azatryptophan” and its abbreviation, “azaTrp,” as used herein, refer to7-azatryptophan.

Modified amino acid residues useful for the optimization of polypeptidesand/or polypeptide compositions of the present invention include, butare not limited to those which are chemically blocked, reversibly orirreversibly, or chemically modified on their N-terminal amino group ortheir side chain groups, or chemically modified in the amide backbone,as for example, N-methylated, D (unnatural amino acids) and L (naturalamino acids) stereoisomers or residues wherein the side chain functionalgroups are chemically modified to another functional group. For example,modified amino acids include without limitation, methionine sulfoxide;methionine sulfone; aspartic acid-(beta-methyl ester), a modified aminoacid of aspartic acid; N-ethylglycine, a modified amino acid of glycine;or alanine carboxamide, and a modified amino acid of alanine. Unnaturalamino acids may be purchased from Sigma-Aldrich (St. Louis, Mo.), Bachem(Torrance, Calif.) or other suppliers. Unnatural amino acids may furtherinclude any of those listed in Table 2 of US patent publication US2011/0172126, the contents of which are incorporated herein by referencein their entirety.

In some embodiments, the amino acid sequences of polypeptides and/orpolypeptide compositions of the present invention may comprise onlynaturally occurring amino acids. While it is known in the art that theterms peptides, polypeptides, and/or fragments thereof imply relativesize, these terms as used herein should not be considered limiting withrespect to the size of the various polypeptide based molecules referredto herein and which are encompassed within this invention, unlessotherwise noted. In some embodiments of the present invention,polypeptides may comprise both naturally and non-naturally occurringand/or modified amino acids or be exclusively comprised of non-naturallyoccurring amino acids.

Polypeptide Variants

According to the present invention, any amino acid based molecule(natural or unnatural) may be termed a “polypeptide” and this termembraces “peptides”, “peptidomimetics” and “proteins.” Polypeptides arealso a category of protein and are traditionally considered to range insize from about 4 to about 50 amino acids. Dipeptides, those having twoamino acid residues, are a category of polypeptide as are tripeptides(polypeptides comprising three amino acids). Polypeptides larger thanabout 50 amino acids are generally termed “proteins.” Polypeptidesequences may be linear or cyclic. For example, a cyclic polypeptide canbe prepared or may result from the formation of disulfide bonds betweentwo cysteine residues in a sequence. A polypeptide can be cyclizedthrough the carboxy terminus, the amino terminus, or through any otherconvenient point of attachment, such as, for example, through the sulfurof a cysteine or any side-chain of an amino acid residue or otherlinkage including, but not limited to, a maleimide linkage, an amidelinkage, an ester linkage, an ether linkage, a thiol ether linkage, ahydrazone linkage, or an acetamide linkage. In some embodiments, cyclicpolypeptides are formed when a molecule acts as a bridging moiety tolink two or more regions of the polypeptide.

The term “amino acid sequence variant” refers to polypeptides with somedifferences in their amino acid sequences as compared to a starting,reference, or native sequence. The amino acid sequence variants maypossess substitutions, deletions, and/or insertions at certain positionswithin the amino acid sequence. Ordinarily, variants will possess atleast about 70% homology to a native or starting sequence, andpreferably, they will be at least about 80%, more preferably at leastabout 90% homologous to a native or starting sequence.

“Homology” as it applies to amino acid sequences is defined as thepercentage of residues in the candidate amino acid sequence that areidentical with the residues in the amino acid sequence of a secondsequence after aligning the sequences and introducing gaps, ifnecessary, to achieve the maximum percent homology. Methods and computerprograms for the alignment are well known in the art. It is understoodthat homology depends on a calculation of percent identity but maydiffer in value due to gaps and penalties introduced in the calculation.

By “homologs” as it applies to amino acid sequences is meant thecorresponding sequence of other species having substantial identity to asecond sequence of a second species.

“Analogs” is meant to include amino acid sequence variants which differby one or more amino acid alterations, e.g., substitutions, additions ordeletions of amino acid residues that still maintain one or more of theproperties of the parent or starting polypeptide.

The present invention contemplates several types of compositions thatinclude polypeptides including variants and derivatives. These includesubstitutional, insertional, deletion and covalent variants andderivatives. The term “derivative” is used synonymously with the term“variant” and refers to a molecule that has been modified or changed inany way relative to a reference molecule or starting molecule.

As such, included within the scope of this invention are polypeptidescontaining substitutions, insertions and/or additions, deletions andcovalent modifications. For example, sequence tags or amino acids, suchas one or more lysines, can be added to the polypeptide sequences of theinvention (e.g., at the N-terminal or C-terminal ends). Sequence tagscan be used for polypeptide purification or localization. Lysines can beused to increase polypeptide solubility or to allow for site specificmodifications, such as, but not limited to, biotinylation or PEGylation.In some cases, polypeptides may be desthiobiotinylated. As used herein,a polypeptide that is desthiobiotinylated may comprise a desthiobiotin(Dtb) moiety conjugated to the epsilon-amino group of a lysine residue.Such lysine residues may be C-terminal residues in some instances.Alternatively, amino acid residues located at the carboxy and aminoterminal regions of the amino acid sequence of a polypeptide mayoptionally be deleted, providing for truncated sequences. Certain aminoacids (e.g., C-terminal or N-terminal residues) may alternatively bedeleted depending on the use of the sequence, as for example, expressionof the sequence as part of a larger sequence, which is soluble, orlinked to a solid support.

“Substitutional variants” when referring to polypeptides are those thathave at least one amino acid residue in a native or starting sequenceremoved and a different amino acid inserted in its place at the sameposition. The substitutions may be single, where only one amino acid inthe molecule has been substituted, or they may be multiple, where two ormore amino acids have been substituted in the same molecule.

As used herein the term “conservative amino acid substitution” refers tothe substitution of an amino acid that is normally present in thesequence with a different amino acid of similar size, charge, orpolarity. Examples of conservative substitutions include thesubstitution of a non-polar (hydrophobic) residue such as isoleucine,valine and leucine for another non-polar residue. Likewise, examples ofconservative substitutions include the substitution of one polar(hydrophilic) residue for another such as between arginine and lysine,between glutamine and asparagine, and between glycine and serine.Additionally, the substitution of a basic residue such as lysine,arginine or histidine for another, or the substitution of one acidicresidue such as aspartic acid or glutamic acid for another acidicresidue are additional examples of conservative substitutions. Examplesof non-conservative substitutions include the substitution of anon-polar (hydrophobic) amino acid residue such as isoleucine, valine,leucine, alanine, methionine for a polar (hydrophilic) residue such ascysteine, glutamine, glutamic acid or lysine and/or a polar residue fora non-polar residue.

“Isosteres” are one of two or more molecules that exhibit somesimilarity of biological properties as a result of having the samenumber of total or valence electrons in the same arrangement and thatconsist of different atoms, not necessarily the same number of atoms.There are two classes of isosteres, classical and non-classical.Classical isosteres have the same number of atoms and/or the same numberof valence electrons whereas non-classical isosteres are molecules thatproduce a similar biological effect in vivo but do not have the samenumber of atoms and/or valence electrons.

According to the present invention, “peptide bond isosteres” are definedas isosteres having properties resembling peptide bonds. Peptide bondisosteres may be of a linear type comprising at least one peptide bondreplacement or may be cyclic and comprise an amine and a carboxylic acidfunction. Such replacement may be with any moiety which improves thephysicochemical, structural or functional properties of the molecule.Replacement of the peptide bond may increase the metabolic stability ofthe polypeptides and reduce or increase the flexibility. Peptide bondisosteres described herein may be mono-, di-, tri-, tetra-, penta-,sexta-, septa-, octa-, nona-, or deca-peptide bond isosteres, meaningthat at least 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 peptidic bonds may bereplaced. Non-limiting examples of linear dipeptide bond isosteres foramide (peptidic) bonds include thioamide, sulfonamide, sulfonate,phosphonamide, phosphonate phosphothioate, phosphinate, alkane, 1 or 2hydroxyethylene, dihydroxylethylene, C—C single bond (alkane), C═Cdouble bond (alkene), C—C triple bond (alkyne), C—O bond (methyleneoxy),O—N or N—O bond, (methylenemino), triazole, hydrazide, urea, ketone,urethane bond, (di)haloalkene, methylenemercapto, methyleneamino,trifluoroethylamino, hydrazide, amideoxy, and others known to those ofskill in the art.

Peptide bond isosteres may also be cyclic molecules that are decoratedwith an amine and a carboxylic acid function. Non-limiting examples ofcyclic peptide bond isosteres with varying ring sizes includecarbacycles, azacycles and oxacycles. Azacycles may be based on analkaloid core which forms a bicyclic structure isostere. An example ofan azacyclic isostere includes an isostere based on a triazole ringformed by a copper catalyzed azide-alkyne cycloaddition. Cyclic peptidebond isosteres described herein may be bi-, tri-, tetra-, penta-sexta-,septa-, octa-nona-deca-peptide cyclic isosteres

“Insertional variants” when referring to polypeptides are those with oneor more amino acids inserted immediately adjacent to an amino acid at aparticular position in a native or starting sequence. “Immediatelyadjacent” to an amino acid means connected to either the alpha-carboxyor alpha-amino functional group of the amino acid.

“Deletional variants” when referring to polypeptides are those with oneor more amino acids in the native or starting amino acid sequenceremoved. Ordinarily, deletional variants will have one or more aminoacids deleted in a particular region of the molecule.

“Truncated variants” when referring to polypeptides are those with oneor more amino acids in the native or starting amino acid sequenceremoved from either terminus of the polypeptide.

According to the present invention, polypeptides may be modified by theaddition of one or more conjugate groups. In some embodiments,polypeptides may be administered in combination with one or moreadditional molecules.

As used herein, a “conjugate” refers to any molecule or moiety appendedto another molecule. In the present invention, conjugates may bepolypeptide (amino acid) based or not. Conjugates may comprise lipids,small molecules, RNA, DNA, polypeptides, polymers, or combinationsthereof. Functionally, conjugates may serve as targeting molecules ormay serve as payload to be delivered to a cell, organ or tissue.Conjugates are typically covalent modifications introduced by reactingtargeted amino acid residues or the termini of the polypeptide with anorganic derivatizing agent that is capable of reacting with selectedside-chains or terminal residues. Such modifications are within theordinary skill in the art and are performed without undueexperimentation.

The conjugation process may involve PEGylation, lipidation,albumination, biotinylation, desthiobiotinylation, the addition of otherpolypeptide tails, or grafting onto antibody Fc domains, CDR regions ofintact antibodies, or antibody domains produced by any number of means.The conjugate may include anchors including cholesterol oleate moiety,cholesteryl laurate moiety, an α-tocopherol moiety, a phytol moiety, anoleate moiety, or an unsaturated cholesterol-ester moiety or alipophilic compound selected from acetanilides, anilides,aminoquinolines, benzhydryl compounds, benzodiazepines, benzofurans,cannabinoids, cyclic polypeptides, dibenzazepines, digitalis glycosides,ergot alkaloids, flavonoids, imidazoles, quinolines, macrolides,naphthalenes, opiates (such as, but not limited to, morphinans or otherpsychoactive drugs), oxazines, oxazoles, phenylalkylamines, piperidines,polycyclic aromatic hydrocarbons, pyrrolidines, pyrrolidinones,stilbenes, sulfonylureas, sulfones, triazoles, tropanes, and vincaalkaloids. Lipidated polypeptides of the invention may includeC-terminally lipidated polypeptides. In some cases, polypeptides arelipidated with saturated or unsaturated C12, C14, C16, C18, or C20.

As used herein, the term “covalent derivative” when referring to apolypeptide includes modification of a native or starting polypeptidewith an organic proteinaceous or non-proteinaceous derivatizing agent,and/or post-translational modification. Covalent modifications aretraditionally introduced by reacting targeted amino acid residues of thepolypeptide with an organic derivatizing agent that is capable ofreacting with selected side-chains or terminal residues, or byharnessing mechanisms of post-translational modifications that functionin selected recombinant host cells. The resultant covalent derivativesare useful in programs directed at identifying residues important forbiological activity, for immunoassays, or for the preparation ofanti-protein antibodies for immunoaffinity purification of therecombinant protein. Such modifications are within the ordinary skill inthe art and are performed without undue experimentation.

Certain post-translational modifications are the result of the action ofrecombinant host cells on an expressed polypeptide. Glutaminyl andasparaginyl residues are frequently post-translationally deamidated tothe corresponding glutamyl and aspartyl residues. Alternatively, theseresidues are deamidated under mildly acidic conditions. Either form ofthese residues may be present in the polypeptides produced in accordancewith the present invention.

Other post-translational modifications include hydroxylation of prolineand lysine, phosphorylation of hydroxyl groups of tyrosinyl, seryl orthreonyl residues, methylation of the alpha-amino groups of lysine,arginine, and histidine side chains (Creighton, T. E., Proteins:Structure and Molecular Properties, W.H. Freeman & Co., San Francisco,1983, pp. 79-86).

Covalent modifications specifically include the bonding ofnon-proteinaceous polymers to polypeptides of the invention.Non-proteinaceous polymers may include a hydrophilic synthetic polymer,i.e., a polymer not otherwise found in nature. However, polymers thatexist in nature and are produced by recombinant or in vitro methods areuseful, as are polymers which are isolated from nature. Hydrophilicpolyvinyl polymers fall within the scope of this invention, e.g.polyvinylalcohol and polyvinylpyrrolidone. The polypeptides may belinked to various non-proteinaceous polymers, such as polyethyleneglycol, polypropylene glycol or polyoxyalkylenes, in the manner setforth in U.S. Pat. Nos. 4,640,835; 4,496,689; 4,301,144; 4,670,417;4,791,192 or 4,179,337.

“Features” when referring to polypeptides are defined as distinct aminoacid sequence-based components of a molecule. Features of thepolypeptide of the present invention include surface manifestations,local conformational shape, folds, loops, half-loops, domains,half-domains, sites, termini or any combination thereof.

As used herein when referring to polypeptides the term “fold” refers tothe resultant conformation of an amino acid sequence upon energyminimization. A fold may occur at the secondary or tertiary level of thefolding process. Examples of secondary level folds include beta sheetsand alpha helices. Examples of tertiary folds include domains andregions formed due to aggregation or separation of physicochemicallydistinct regions. Regions formed in this way include hydrophobic andhydrophilic pockets, and the like.

As used herein the term “turn” as it relates to protein conformationmeans a bend which alters the direction of the backbone of a polypeptideand may involve one, two, three or more amino acid residues.

As used herein when referring to polypeptides the term “loop” refers toa structural feature of a polypeptide which may serve to reverse thedirection of the backbone of a polypeptide. Where the loop is found in apolypeptide and only alters the direction of the backbone, it maycomprise four or more amino acid residues. Oliva et al. have identifiedat least 5 classes of protein loops (Oliva, B. et al., J Mol Biol. 1997Mar. 7; 266(4):814-30; the contents of which are herein incorporated byreference in their entirety). Loops may be open or closed. Closed loopsor “cyclic” loops may be formed when two amino acids are connected by abridging moiety. The cyclic loop comprises the amino acids along thepolypeptide present between the bridged amino acids. Cyclic loops maycomprise 2, 3, 4, 5, 6, 7, 8, 9, 10 or more amino acids.

As used herein when referring to polypeptides the term “half-loop”refers to a portion of an identified loop having at least half thenumber of amino acid resides as the loop from which it is derived. It isunderstood that loops may not always contain an even number of aminoacid residues. Therefore, in those cases where a loop contains or isidentified to comprise an odd number of amino acids, a half-loop of theodd-numbered loop will comprise the whole number portion or next wholenumber portion of the loop (number of amino acids of the loop/2+/−0.5amino acids). For example, a loop identified as a 7 amino acid loopcould produce half-loops of 3 amino acids or 4 amino acids(7/2=3.5+/−0.5 being 3 or 4).

As used herein when referring to proteins, the term “region” refers to azone or general area. In some embodiments, when referring to a protein,a region may comprise a linear sequence of amino acids along the proteinor may comprise a specific secondary or tertiary structure and/or one ormore features or protein domains.

As used herein, the term “domain,” when referring to proteins, refers toa motif of a polypeptide having one or more identifiable structural(such as secondary or tertiary structures) or functional characteristicsor properties (e.g., binding capacity, serving as a site forprotein-protein interactions.)

As used herein, the term “half-domain,” when referring to proteins,refers to a portion of an identified domain having at least half thenumber of amino acid resides as the domain from which it is derived. Itis understood that domains may not always contain an even number ofamino acid residues. Therefore, in those cases where a domain containsor is identified to comprise an odd number of amino acids, a half-domainof the odd-numbered domain will comprise the whole number portion ornext whole number portion of the domain (number of amino acids of thedomain/2+/−0.5 amino acids). For example, a domain identified as a 7amino acid domain could produce half-domains of 3 amino acids or 4 aminoacids (7/2=3.5+/−0.5 being 3 or 4). It is also understood thatsub-domains may be identified within domains or half-domains, thesesubdomains possessing less than all of the structural or functionalproperties identified in the domains or half domains from which theywere derived. It is also understood that the amino acids that compriseany of the domain types herein need not be contiguous along the backboneof the polypeptide (i.e., nonadjacent amino acids may fold structurallyto produce a domain, half-domain or subdomain).

As used herein when referring to polypeptides the term “site” as itpertains to amino acid based embodiments is used synonymously with“amino acid residue” and “amino acid side chain.” A site represents aposition within a polypeptide that may be modified, manipulated,altered, derivatized or varied within the polypeptide based molecules ofthe present invention.

As used herein the terms “termini” or “terminus” when referring topolypeptides refers to an extremity of a polypeptide. Such extremity isnot limited only to the first or final site of the polypeptide but mayinclude additional amino acids in the terminal regions. The polypeptidebased molecules of the present invention may be characterized as havingboth an N-terminus and a C-terminus. Polypeptides and/or polypeptidecompositions of the present invention are in some cases made up ofmultiple polypeptide chains brought together by disulfide bonds or bynon-covalent forces (multimers, oligomers). These sorts of proteins willhave multiple N- and C-termini. Alternatively, the termini of thepolypeptides may be modified such that they begin or end, as the casemay be, with a non-polypeptide based moiety such as an organicconjugate.

In one embodiment, polypeptides of the present invention may include aterminal region. As used herein, “terminal region” is a terminal regionof amino acids that may include a cysteine. The terminal region may bean N- and/or a C-terminal region. In some embodiments, terminal regionsmay be connected to the parent polypeptides using a bridging moiety. Asused herein, “parent polypeptide” refers to the part of the polypeptidethat does not include the terminal region. The terminal region may beseparated from the parent polypeptide by 1, 2, 3, 4, 5, 6, 7, 8, 9, 10or more residues. The residues added may be selected from, but are notlimited to, any natural or unnatural amino acid, the N-methylated formof any natural or unnatural amino acid, the D-stereoisomer of anynatural or unnatural amino acid, norvaline, tert-butylglycine,phenylglycine, azatryptophan, 7-azatryptophan, 4-fluorophenylalanine,penicillamine, sarcosine, homocysteine, 1-aminocyclopropanecarboxylicacid, 1-aminocyclobutanecarboxylic acid, 1-aminocyclopentanecarboxylicacid, 1-aminocyclohexanecarboxylic acid,4-aminotetrahydro-2H-pyran-4-carboxylic acid, aminoisobutyric acid,(S)-2-amino-3-(1H-tetrazol-5-yl)propanoic acid, cyclopentylglycine,cyclohexylglycine, cyclopropylglycine, f-co-methyl-arginine,4-chlorophenylalanine, 3-chlorotyrosine, 3-fluorotyrosine,5-fluorotryptophan, 5-chlorotryptophan, citrulline,4-chloro-homophenylalanine, homophenylalanine,4-aminomethyl-phenylalanine, 3-aminomethyl-phenylalanine, octylglycine,norleucine, tranexamic acid, 2-amino pentanoic acid, 2-amino hexanoicacid, 2-amino heptanoic acid, 2-amino octanoic acid, 2-amino nonanoicacid, 2-amino decanoic acid, 2-amino undecanoic acid, 2-amino dodecanoicacid, aminovaleric acid, and 2-(2-aminoethoxy)acetic acid, pipecolicacid, 2-carboxy azetidine, hexafluoroleucine, 3-Fluorovaline,2-amino-4,4-difluoro-3-methylbutanoic acid, 3-fluoro-isoleucine,4-fluoroisoleucine, 5-fluoroisoleucine, 4-methyl-phenylglycine,4-ethyl-phenylglycine, 4-isopropyl-phenylglycine,(S)-2-amino-5-(3-methylguanidino) pentanoic acid,(S)-2-amino-3-(4-(aminomethyl)phenyl)propanoic acid,(S)-2-amino-3-(3-(aminomethyl)phenyl)propanoic acid,(S)-2-amino-4-(2-aminobenzo[d]oxazol-5-yl)butanoic acid, (S)-leucinol,(S)-valinol, (S)-tert-leucinol, (R)-3-methylbutan-2-amine,(S)-2-methyl-1-phenylpropan-1-amine, and(S)—N,2-dimethyl-1-(pyridin-2-yl)propan-1-amine,(S)-2-amino-3-(oxazol-2-yl)propanoic acid,(S)-2-amino-3-(oxazol-5-yl)propanoic acid,(S)-2-amino-3-(1,3,4-oxadiazol-2-yl)propanoic acid,(S)-2-amino-3-(1,2,4-oxadiazol-3-yl)propanoic acid,(S)-2-amino-3-(5-fluoro-1H-indazol-3-yl)propanoic acid, and(S)-2-amino-3-(1H-indazol-3-yl)propanoic acid.

Additional unnatural amino acids that are useful in the optimization ofpolypeptides of the invention include but are not limited to fluorinatedaminoacids wherein one or more carbon bound hydrogen atoms are replacedby fluorine. The number of fluorine atoms included can range from 1 upto and including all of the hydrogen atoms. Examples of such amino acidsinclude but are not limited to 3-fluoroproline, 3,3-difluoroproline,4-fluoroproline, 4,4-difluoroproline, 3,4-difluroproline,3,3,4,4-tetrafluoroproline, 4-fluorotryptophan, 6-fluorotryptophan,7-fluorotryptophan, and stereoisomers thereof.

Further unnatural amino acids that are useful in the optimization ofpolypeptides of the invention include but are not limited to those thatare disubstituted at the α-carbon. These include amino acids in whichthe two substituents on the α-carbon are the same, for example α-aminoisobutyric acid, and 2-amino-2-ethyl butanoic acid, as well as thosewhere the substituents are different, for example α-methylphenylglycineand α-methylproline. Further the substituents on the α-carbon may betaken together to form a ring, for example 1-aminocyclopentanecarboxylicacid, 1-aminocyclobutanecarboxylic acid, 1-aminocyclohexanecarboxylicacid, 3-aminotetrahydrofuran-3-carboxylic acid,3-aminotetrahydropyran-3-carboxylic acid,4-aminotetrahydropyran-4-carboxylic acid,3-aminopyrrolidine-3-carboxylic acid, 3-aminopiperidine-3-carboxylicacid, 4-aminopiperidinnne-4-carboxylix acid, and stereoisomers thereof.

Additional unnatural amino acids that are useful in the optimization ofpolypeptides of the invention include but are not limited to analogs oftryptophan in which the indole ring system is replaced by another 9 or10 membered bicyclic ring system comprising 0, 1, 2, 3, or 4 heteroatomsindependently selected from N,O, or S. Each ring system may besaturated, partially unsaturated or fully unsaturated. The ring systemmay be substituted by 0, 1, 2, 3, or 4 substituents at any substitutableatom. Each substituent is independently selected from H, F, Cl, Br, CN,oxo, COOR, CONRR′, OR, NRR′. Each R and R′ is independently selectedfrom H, C1-C20 alkyl, C1-C20 alkyl-O—C1-20 alkyl.

In some embodiments, analogs of tryptophan (also referred to herein as“tryptophan analogs”) that are useful in the optimization ofpolypeptides of the invention include, but are not limited to5-fluorotryptophan [(5-F)W], 5-methyl-O-tryptophan [(5-MeO)W],1-methyltryptophan [(1-Me-W) or (1-Me)W], D-tryptophan (D-Trp),7-azatryptophan (including, but not limited to 4-azatryptophan,7-azatryptophan and 5-azatryptophan,) 5-chlorotryptophan,4-fluorotryptophan, 6-fluorotryptophan, 7-fluorotryptophan, andstereoisomers thereof. Except where indicated to the contrary, the term“azatryptophan” and its abbreviation, “azaTrp,” as used herein, refer to7-azatryptophan.

In one embodiment, polypeptides of the present invention may include aterminal modification at the N- or C-termini with the addition of 1, 2,3, 4, 5, 6, 7, 8, 9, 10 or more residues and/or a cysteine in theterminal region. The residues added may be selected from, but are notlimited to, any natural or unnatural amino acid, the N-methylated formof any natural or unnatural amino acid, the D-stereoisomer of any aminoacid, norvaline, tert-butylglycine, phenylglycine, azatryptophan,7-azatryptophan, 4-fluorophenylalanine, penicillamine, sarcosine,homocysteine, 1-aminocyclopropanecarboxylic acid,1-aminocyclobutanecarboxylic acid, 1-aminocyclopentanecarboxylic acid,1-aminocyclohexanecarboxylic acid,4-aminotetrahydro-2H-pyran-4-carboxylic acid, aminoisobuteric acid,(S)-2-amino-3-(1H-tetrazol-5-yl)propanoic acid, cyclopentylglycine,cyclohexylglycine, cyclopropylglycine, r-co-methyl-arginine,4-chlorophenylalanine, 3-chlorotyrosine, 3-fluorotyrosine,5-fluorotryptophan, 5-chlorotryptophan, citrulline,4-chloro-homophenylalanine, homophenylalanine,4-aminomethyl-phenylalanine, 3-aminomethyl-phenylalanine, octylglycine,norleucine, tranexamic acid, 2-amino pentanoic acid, 2-amino hexanoicacid, 2-amino heptanoic acid, 2-amino octanoic acid, 2-amino nonanoicacid, 2-amino decanoic acid, 2-amino undecanoic acid, 2-amino dodecanoicacid, aminovaleric acid, and 2-(2-aminoethoxy)acetic acid, pipecolicacid, 2-carboxy azetidine, hexafluoroleucine, 3-Fluorovaline,2-amino-4,4-difluoro-3-methylbutanoic acid, 3-fluoro-isoleucine,4-fluoroisoleucine, 5-fluoroisoleucine, 4-methyl-phenylglycine,4-ethyl-phenylglycine, 4-isopropyl-phenylglycine,(S)-2-amino-5-(3-methylguanidino) pentanoic acid,(S)-2-amino-3-(4-(aminomethyl)phenyl)propanoic acid,(S)-2-amino-3-(3-(aminomethyl)phenyl)propanoic acid,(S)-2-amino-4-(2-aminobenzo[d]oxazol-5-yl)butanoic acid, (S)-leucinol,(S)-valinol, (S)-tert-leucinol, (R)-3-methylbutan-2-amine,(S)-2-methyl-1-phenylpropan-1-amine, and(S)—N,2-dimethyl-1-(pyridin-2-yl)propan-1-amine,(S)-2-amino-3-(oxazol-2-yl)propanoic acid,(S)-2-amino-3-(oxazol-5-yl)propanoic acid,(S)-2-amino-3-(1,3,4-oxadiazol-2-yl)propanoic acid,(S)-2-amino-3-(1,2,4-oxadiazol-3-yl)propanoic acid,(S)-2-amino-3-(5-fluoro-1H-indazol-3-yl)propanoic acid, and(S)-2-amino-3-(1H-indazol-3-yl)propanoic acid.

Polypeptides of the present invention may be conjugated to a polypeptidethat increases or decreases plasma protein binding including but notlimited to those described in Dennis, M. S. et al., Albumin binding as ageneral strategy for improving the pharmacokinetics of proteins. J BiolChem. 2002 Sep. 20; 277(38):35035-43; Nguyen, A. et al., Thepharmacokinetics of an albumin-binding Fab (AB Fab) can be modulated asa function of affinity for albumin. Protein Eng Des Sel. 2006 July;19(7):291-7 and Langerheim, J. F. et al., Improving thepharmacokinetics/pharmacodynamics of prolactin, GH, and theirantagonists by fusion to a synthetic albumin-binding polypeptide. JEndocrinol. 2009 December; 203(3):375-87. In some embodiments, suchpolypeptides bind serum albumin (referred to herein as “albumin-bindingpolypeptides”). In some embodiments, albumin-binding polypeptides arecyclized by disulfide bond formation between cysteine residues presentin their polypeptide sequences. In some embodiments, albumin-bindingpolypeptides are conjugated by either their N or C-terminal ends. Insome embodiments, conjugation to an albumin-binding polypeptidemodulates the amount of time that a polypeptide of the present inventionremains intact in a subject. In a preferred embodiment, conjugation toan albumin-binding polypeptide increases the amount of time that apolypeptide of the present invention remains in the blood of a subject.Polypeptides of the present invention may be conjugated to polypeptidesthat have cell penetrating properties (referred to herein as “cellpenetrating polypeptides”) including but not limited those disclosed inMilletti, F., Cell-penetrating peptides: classes, origin, and currentlandscape. Drug Discov Today. 2012 August; 17(15-16):850-60. Additionalcell penetrating polypeptides are known to those skilled in the art.Polypeptides of the present invention may be conjugated to any of thepolypeptide conjugates taught, for example, in US patent publicationsUS20110172126 or US20030040472 the contents of which are incorporatedherein by reference in their entirety. Polypeptides of the presentinvention may be conjugated to a lipophilic molecule that increasesplasma protein binding such as the liphophilic substituents taught, forexample, in U.S. Pat. No. 6,268,343 or US Publication No.US2013/0053311, the contents of each of which are herein incorporated byreference in their entirety.

Once any of the features have been identified or defined as a desiredcomponent of a polypeptide, any of several manipulations and/ormodifications of these features may be performed by moving, swapping,inverting, deleting, randomizing or duplicating. Furthermore, it isunderstood that manipulation of features may result in the same outcomeas a modification to the molecules of the invention. For example, amanipulation which involved deleting a domain would result in thealteration of the length of a molecule just as modification of a nucleicacid to encode less than a full length molecule would.

Modifications and manipulations can be accomplished by methods known inthe art such as, but not limited to, site directed mutagenesis. Theresulting modified molecules may then be tested for activity using invitro or in vivo assays such as those described herein or any othersuitable screening assay known in the art.

According to the present invention, the polypeptides may comprise aconsensus sequence which is discovered through rounds ofexperimentation. As used herein a “consensus” sequence is a singlesequence which represents a collective population of sequences allowingfor variability at one or more sites.

The term “identity” as known in the art, refers to a relationshipbetween the sequences of two or more polypeptides, as determined bycomparing the sequences. In the art, identity also means the degree ofsequence relatedness between polypeptides, as determined by the numberof matches between strings of two or more amino acid residues. Identitymeasures the percent of identical matches between the smaller of two ormore sequences with gap alignments (if any) addressed by a particularmathematical model or computer program (i.e., “algorithms”). Identity ofrelated polypeptides can be readily calculated by known methods. Suchmethods include, but are not limited to, those described previously byothers (Lesk, A. M., ed., Computational Molecular Biology, OxfordUniversity Press, New York, 1988; Smith, D. W., ed., Biocomputing:Informatics and Genome Projects, Academic Press, New York, 1993;Griffin, A. M. et al., ed., Computer Analysis of Sequence Data, Part 1,Humana Press, New Jersey, 1994; von Heinje, G., Sequence Analysis inMolecular Biology, Academic Press, 1987; Gribskov, M. et al., ed.,Sequence Analysis Primer, M. Stockton Press, New York, 1991; and Carilloet al., Applied Math, SIAM J, 1988, 48, 1073).

In some embodiments, a polypeptide variant may have the same or asimilar activity as the reference polypeptide. Alternatively, a variantmay have an altered activity (e.g., increased or decreased) relative toa reference polypeptide. Generally, variants of a particular polypeptideof the invention will have at least about 40%, 45%, 50%, 55%, 60%, 65%,70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% butless than 100% sequence identity to that of a particular referencepolypeptide as determined by sequence alignment programs and parametersdescribed herein and known to those skilled in the art. Such tools foralignment include those of the BLAST suite (Altschul, S. F. et al.,Gapped BLAST and PSI-BLAST: a new generation of protein database searchprograms, Nucleic Acids Res. 1997, 25:3389-3402) Other tools aredescribed herein, specifically in the definition of“identity.”

Default parameters in the BLAST algorithm include, for example, anexpected threshold of 10, Word size of 28, Match/Mismatch Scores 1, −2,Gap costs Linear. Any filter can be applied as well as a selection forspecies specific repeats, e.g., Homo sapiens.

Abbreviations Used in Polypeptides

As used herein, abbreviations have the following meaning: “Ac” and “NH2”indicate acetyl and amidated termini, respectively; “Nvl” stands fornorvaline; “Phg” stands for phenylglycine; “Tbg” stands fortert-butylglycine; “Chg” stands for cyclohexylglycine; “(N-Me)X” standsfor the N-methylated form of the amino acid indicated by the letter orthree letter amino acid code in place of variable “X” written asN-methyl-X [e.g. (N-Me)A or (N-Me)Ala stand for the N-methylated form ofalanine or N-methyl-alanine]; “azaTrp” stands for azatryptophan;“(4-F)Phe” stands for 4-fluorophenylalanine; “Tyr(OMe)” stands forO-methyl tyrosine, “Aib” stands for amino isobutyric acid; “(homo)F” or“(homo)Phe” stands for homophenylalanine; “(2-OMe)Phg” refers to2-O-methylphenylglycine; “(5-F)W” refers to 5-fluorotryptophan; “D-X”refers to the D-stereoisomer of the given amino acid “X” [e.g. (D-Chg)stands for D-cyclohexylglycine]; “(5-MeO)W” refers to5-methyl-O-tryptophan; “homoC” refers to homocysteine; “(1-Me-W)” or“(1-Me)W” refers to 1-methyltryptophan; “Nle” refers to norleucine;“Tiq” refers to a tetrahydroisoquinoline residue; “Asp(T)” refers to(S)-2-amino-3-(1H-tetrazol-5-yl)propanoic acid; “(3-Cl-Phe)” refers to3-chlorophenylalanine; “[(N-Me-4-F)Phe]” or “(N-Me-4-F)Phe” refers toN-methyl-4-fluorophenylalanine; “(m-Cl-homo)Phe” refers to meta-chlorohomophenylalanine; “(des-amino)C” refers to 3-thiopropionic acid;“(alpha-methyl)D” refers to alpha-methyl L-aspartic acid; “2Nal” refersto 2-naphthylalanine; “(3-aminomethyl)Phe” refers to3-aminomethyl-L-phenyalanine; “Cle” refers to cycloleucine; “Ac-Pyran”refers to 4-amino-tetrahydro-pyran-4-carboxylic acid; “(Lys-C16)” refersto N-ε-palmitoyl lysine; “(Lys-C12)” refers to N-ε-lauryl lysine;“(Lys-C10)” refers to N-ε-capryl lysine; “(Lys-C8)” refers toN-ε-caprylic lysine; “[xXylyl(y, z)]” refers to the xylyl bridgingmoiety between two thiol containing amino acids where x may be m, p or oto indicate the use of meta-, para- or ortho-dibromoxylenes(respectively) to generate bridging moieties and the numericalidentifiers, y and z, place the amino acid position within thepolypeptide of the amino acids participating in the cyclization;“[cyclo(y,z)]” refers to the formation of a bond between two amino acidresidues where the numerical identifiers, y and z, place the position ofthe residues participating in the bond; “[cyclo-olefinyl(y,z)]” refersto the formation of a bond between two amino acid residues by olefinmetathesis where the numerical identifiers, y and z, place the positionof the residues participating in the bond; “[cyclo-thioalkyl(y,z)]”refers to the formation of a thioether bond between two amino acidresidues where the numerical identifiers, y and z, place the position ofthe residues participating in the bond; “[cyclo-triazolyl(y,z)]” refersto the formation of a triazole ring between two amino acid residueswhere the numerical identifiers, y and z, place the position of theresidues participating in the bond. “B20” refers to N-ε-(PEG2-γ-glutamicacid-N-α-octadecanedioic acid) lysine [also known as(1S,28S)-1-amino-7,16,25,30-tetraoxo-9,12,18,21-tetraoxa-6,15,24,29-tetraazahexatetracontane-1,28,46-tricarboxylicacid.]

B20

“B28” refers to N-ε-(PEG24-γ-glutamic acid-N-α-hexadecanoyl)lysine.

B28

“K14” refers toN-ε-1-(4,4-dimethyl-2,6-dioxocyclohex-1-ylidene)-3-methylbutyl-L-lysine.All other symbols refer to the standard one-letter amino acid code.

Antibodies

In some embodiments, compounds and/or compositions of the presentinvention may comprise antibodies or fragments thereof. As used herein,the term “antibody” is referred to in the broadest sense andspecifically covers various embodiments including, but not limited tomonoclonal antibodies, polyclonal antibodies, multispecific antibodies(e.g. bispecific antibodies formed from at least two intact antibodies),and antibody fragments such as diabodies so long as they exhibit adesired biological activity. Antibodies of the present invention mayalso comprise human antibodies or humanized antibodies. Antibodies areprimarily amino-acid based molecules but may also comprise one or moremodifications (including, but not limited to the addition of sugarmoieties, fluorescent moieties, chemical tags, etc.)

As used herein the term, “antibody fragment” refers to any portion of anintact antibody. In some embodiments, antibody fragments compriseantigen binding regions from intact antibodies. Examples of antibodyfragments may include, but are not limited to Fab, Fab′, F(ab′)2, and Fvfragments; diabodies; linear antibodies; single-chain antibodymolecules; and multispecific antibodies formed from antibody fragments.Papain digestion of antibodies produces two identical antigen-bindingfragments, called “Fab” fragments, each with a single antigen-bindingsite. Also produced is a residual “Fc” fragment, whose name reflects itsability to crystallize readily. Pepsin treatment yields an F(ab′)₂fragment that has two antigen-binding sites and is still capable ofcross-linking antigen. Compounds and/or compositions of the presentinvention may comprise one or more of these fragments. For the purposesherein, an “antibody” may comprise a heavy and light variable domain aswell as an Fc region.

As used herein, the term “native antibody” refers to a usuallyheterotetrameric glycoprotein of about 150,000 daltons, composed of twoidentical light (L) chains and two identical heavy (H) chains. Eachlight chain is linked to a heavy chain by one covalent disulfide bond,while the number of disulfide linkages varies among the heavy chains ofdifferent immunoglobulin isotypes. Each heavy and light chain also hasregularly spaced intrachain disulfide bridges. Each heavy chain has atone end a variable domain (V_(H)) followed by a number of constantdomains. Each light chain has a variable domain at one end (V_(L)) and aconstant domain at its other end; the constant domain of the light chainis aligned with the first constant domain of the heavy chain, and thelight chain variable domain is aligned with the variable domain of theheavy chain.

As used herein, the term “variable domain” refers to specific antibodydomains that differ extensively in sequence among antibodies and areused in the binding and specificity of each particular antibody for itsparticular antigen.

As used herein, the term “Fv” refers to antibody fragments comprisingcomplete antigen-recognition and antigen-binding sites. These regionsconsist of a dimer of one heavy chain and one light chain variabledomain in tight, non-covalent association.

As used herein, the term “light chain” refers to a component of anantibody from any vertebrate species assigned to one of two clearlydistinct types, called kappa and lambda based on amino acid sequences ofconstant domains.

Depending on the amino acid sequence of the constant domain of theirheavy chains, antibodies can be assigned to different classes. There arefive major classes of intact antibodies: IgA, IgD, IgE, IgG, and IgM,and several of these may be further divided into subclasses (isotypes),e.g., IgG1, IgG2, IgG3, IgG4, IgA, and IgA2.

As used herein, the term “Single-chain Fv” or “scFv” refers to a fusionprotein of V_(H) and V_(L) antibody domains, wherein these domains arelinked together into a single polypeptide chain. In some embodiments,the Fv polypeptide linker enables the scFv to form the desired structurefor antigen binding.

As used herein, the term “diabody” refers to a small antibody fragmentwith two antigen-binding sites. Diabodies comprise a heavy chainvariable domain V_(H) connected to a light chain variable domain V_(L)in the same polypeptide chain. By using a linker that is too short toallow pairing between the two domains on the same chain, the domains areforced to pair with the complementary domains of another chain andcreate two antigen-binding sites. Diabodies are described more fully in,for example, EP 404,097; WO 93/11161; and Hollinger et al. (Hollinger,P. et al., “Diabodies”:Small bivalent and bispecific antibody fragments.PNAS. 1993. 90:6444-8) the contents of each of which are incorporatedherein by reference in their entirety.

As used herein, the term “monoclonal antibody” refers to an antibodyobtained from a population of substantially homogeneous cells (orclones), i.e., the individual antibodies comprising the population areidentical and/or bind the same epitope, except for possible variantsthat may arise during production of the monoclonal antibodies, suchvariants generally being present in minor amounts. In contrast topolyclonal antibody preparations that typically include differentantibodies directed against different determinants (epitopes), eachmonoclonal antibody is directed against a single determinant on theantigen

The modifier “monoclonal” indicates the character of the antibody asbeing obtained from a substantially homogeneous population ofantibodies, and is not to be construed as requiring production of theantibody by any particular method. The monoclonal antibodies hereininclude “chimeric” antibodies (immunoglobulins) in which a portion ofthe heavy and/or light chain is identical with or homologous tocorresponding sequences in antibodies derived from a particular speciesor belonging to a particular antibody class or subclass, while theremainder of the chain(s) is identical with or homologous tocorresponding sequences in antibodies derived from another species orbelonging to another antibody class or subclass, as well as fragments ofsuch antibodies.

As used herein, the term “humanized antibody” refers to a chimericantibody comprising a minimal portion from one or more non-human (e.g.,murine) antibody source with the remainder derived from one or morehuman immunoglobulin sources. For the most part, humanized antibodiesare human immunoglobulins (recipient antibody) in which residues fromthe hypervariable region from an antibody of the recipient are replacedby residues from the hypervariable region from an antibody of anon-human species (donor antibody) such as mouse, rat, rabbit ornonhuman primate having the desired specificity, affinity, and/orcapacity.

As used herein, the term “hypervariable region” refers to regions withinthe antigen binding domain of an antibody comprising amino acid residuesresponsible for antigen binding. The amino acids present within thehypervariable regions determine the structure of the complementaritydetermining region (CDR). As used herein, the term “CDR” refers toregions of antibodies comprising a structure that is complimentary toits target antigen or epitope.

In some embodiments, compounds and/or compositions of the presentinvention may be or comprise antibody mimetics. As used herein, the term“antibody mimetic” refers to any molecule which mimics the function oreffect of an antibody and which binds specifically and with highaffinity to their molecular targets. In some embodiments, antibodymimetics may be monobodies, designed to incorporate the fibronectin typeIII domain (Fn3) as a protein scaffold (U.S. Pat. No. 6,673,901 and U.S.Pat. No. 6,348,584, the contents of each of which are hereinincorporated by reference in their entirety). In some embodiments,antibody mimetics may include those known in the art including, but notlimited to affibody molecules, affilins, affitins, anticalins, avimers,Centyrins, DARPINS™, Fynomers, Adnectins, and Kunitz domain peptides. Inother embodiments, antibody mimetics may include one or more non-peptideregion.

As used herein, the term “antibody variant” refers to a biomoleculeresembling an antibody in structure and/or function comprising somedifferences in their amino acid sequence, composition or structure ascompared to a native antibody.

The preparation of antibodies, whether monoclonal or polyclonal, isknown in the art. Techniques for the production of antibodies are wellknown in the art and described, e.g. in Harlow and Lane “Antibodies, ALaboratory Manual”, Cold Spring Harbor Laboratory Press, 1988 and Harlowand Lane “Using Antibodies: A Laboratory Manual” Cold Spring HarborLaboratory Press, 1999.

In some embodiments, polypeptide sequences provided herein may beutilized in the production of one or more antibodies. In some cases,such polypeptide sequences may be incorporated into antibody variabledomains. Such variable domains may be incorporated into antibodies,antibody mimetics or antibody variants.

Small Molecules

In some embodiments, compounds of the present invention may be smallmolecules. Such compounds may comprise a size from about 100 to about2000 Daltons (e.g. from about 100 to about 200, to about 300, to about400, to about 500, to about 600, to about 700, to about 800, to about900, to about 1000, to about 1100, to about 1200, to about 1300, toabout 1400, to about 1500, to about 1600, to about 1700, to about 1800,to about 1900 or to about 2000 Daltons.) Small molecules may benon-peptidic or share some or many characteristics of polypeptides andcyclic polypeptides, including amide bonds, cyclic structures, and aminoacid-like substituents.

Aptamers

In some embodiments, compounds of the present invention may compriseaptamers (Keefe, A. D., Pai, S. and Ellington, A. (2010). Nat. Rev. DrugDiscovery 9:537-550). As used herein, the term “aptamer” refers tooligonucleic or polypeptide molecules that are capable of bindingspecific target molecules. Some aptamers may adopt a three-dimensionalconformation capable of binding such target molecules with high affinityand specificity.

Isotopic Variations

Polypeptides of the present invention may comprise one or more atomsthat are isotopes. As used herein, the term “isotope” refers to achemical element that has one or more additional neutrons. In oneembodiment, polypeptides of the present invention may be deuterated. Asused herein, the term “deuterated” refers to a substance that has hadone or more hydrogen atoms replaced by deuterium isotopes. Deuteriumisotopes are isotopes of hydrogen. The nucleus of hydrogen contains oneproton while deuterium nuclei contain both a proton and a neutron.Compounds and pharmaceutical compositions of the present invention maybe deuterated in order to change a physical property, such as stability,or to allow them to be used in diagnostic and experimental applications.

Formulation and Delivery

The term “pharmaceutical composition” refers to a composition comprisingat least one active ingredient (e.g., such as a polypeptide) in a formand amount that permits the active ingredient to be therapeuticallyeffective.

Polypeptide formulations of the present invention include controlledduodenal release formulations, time release formulations,osmotic-controlled release delivery systems, microemulsions,microspheres, liposomes, nanoparticles, patches, pumps, drug depots, andthe like. Specifically included in the present invention are solid oraldosage forms, such as powders, softgels, gelcaps, capsules, pills, andtablets.

The pharmaceutical compositions of the present invention may beadministered by any route that results in a therapeutically effectiveoutcome. These include, but are not limited to enteral, gastroenteral,epidural, oral, peridural, intracerebral (into the cerebrum),intratracheal (into the airways for delivery to the lung),intracerebroventricular (into the cerebral ventricles), epicutaneous(application onto the skin), intradermal, (into the skin itself),subcutaneous (under the skin), nasal administration (through the nose),intravenous (into a vein), intraarterial (into an artery), intramuscular(into a muscle), intracardiac (into the heart), intraosseous infusion(into the bone marrow), intrathecal (into the spinal canal),intraperitoneal, (infusion or injection into the peritoneum),intravesical infusion, intravitreal, (into the posterior chamber of theeye), intracavernous injection, (into the base of the penis),intravaginal administration, intrauterine, extra-amnioticadministration, transdermal (diffusion through the intact skin forsystemic distribution), transmucosal (diffusion through a mucousmembrane), insufflation (snorting), buccal, sublingual, sublabial,enema, eye drops (onto the conjunctiva), or in ear drops.

In some embodiments, polypeptides of the present invention areformulated in a sterile aqueous solution. In some embodiments,polypeptides of the present invention are formulated in a lipid ornon-lipid formulation. In another embodiment, polypeptides of thepresent invention are formulated in a cationic or non-cationic lipidformulation. In either embodiment, the sterile aqueous solution maycontain additional active or inactive components. Inactive components,also referred to herein as “excipients,” can include, but are notlimited to, physiologically compatible salts, sugars, bulking agents,surfactants, or buffers.

Polypeptides and/or polypeptide compositions of the present inventionmay comprise or be formulated or delivered in conjunction with one ormore carrier agents. As used herein, the term “carrier” refers to asubstance that aids in the delivery or improves the effectiveness of thepolypeptides and/or polypeptide compositions of the present invention.The carrier agent can be a naturally occurring substance, such as aprotein (e.g., human serum albumin (HSA), low-density lipoprotein (LDL),or globulin); carbohydrate (e.g., a dextran, pullulan, chitin, chitosan,inulin, cyclodextrin, or hyaluronic acid); or lipid. The carriermolecule can also be a recombinant or synthetic molecule, such as asynthetic polymer, e.g., a synthetic polyamino acid. Examples ofpolyamino acids include poly-L-lysine (PLL), poly-L-aspartic acid and,poly-L-glutamic acid, as well as polymers comprising the D-stereoisomersof these amino acids. Other carriers includepoly(L-lactide-co-glycolide) copolymer, polyethylene glycol (PEG),polyvinyl alcohol (PVA), poly(2-ethylacryllic acid), andN-isopropylacrylamide polymers. Other useful carrier molecules can beidentified by routine methods.

In some embodiments, compounds of the present invention may be combinedwith one or more pharmaceutically acceptable excipient to form apharmaceutical composition. As used herein, the term “pharmaceuticallyacceptable” refers to those compounds, materials, compositions, and/ordosage forms which are, within the scope of sound medical judgment,suitable for use in contact with the tissues of human beings and animalswithout excessive toxicity, irritation, allergic response, or otherproblem or complication, commensurate with a reasonable benefit/riskratio. The phrase “pharmaceutically acceptable excipient,” as usedherein, refers any ingredient other than the inventive compoundsdescribed herein (for example, a vehicle capable of suspending ordissolving the active compound) and having the properties of beingsubstantially nontoxic and non-inflammatory in a patient. Excipients mayinclude, for example: antiadherents, antioxidants, binders, coatings,compression aids, disintegrants, dyes (colors), emollients, emulsifiers,fillers (diluents), film formers or coatings, flavors, fragrances,glidants (flow enhancers), lubricants, preservatives, printing inks,sorbents, suspensing or dispersing agents, sweeteners, and waters ofhydration. Exemplary excipients include, but are not limited to:butylated hydroxytoluene (BHT), calcium carbonate, calcium phosphate(dibasic), calcium stearate, croscarmellose, crosslinked polyvinylpyrrolidone, citric acid, crospovidone, cysteine, ethylcellulose,gelatin, hydroxypropyl cellulose, hydroxypropyl methylcellulose,lactose, magnesium stearate, maltitol, mannitol, methionine,methylcellulose, methyl paraben, microcrystalline cellulose,polyethylene glycol, polyvinyl pyrrolidone, povidone, pregelatinizedstarch, propyl paraben, retinyl palmitate, shellac, silicon dioxide,sodium carboxymethyl cellulose, sodium citrate, sodium starch glycolate,sorbitol, starch (corn), stearic acid, sucrose, talc, titanium dioxide,vitamin A, vitamin E, vitamin C, and xylitol. In some embodiments,pharmaceutical compositions comprise one or more active polypeptideingredients together with ethanol, corn oil-mono-di-triglycerides,hydrogenated castor oil, DL-tocopherol, propylene glycol, gelatin,glycerol, colorants, flavors and sweeteners.

In other embodiments, pharmaceutical compositions comprise one or moreactive polypeptide ingredients together with a delivery agent such as4-(2-hydroxy-4-methoxybenzamido)butanoic acid (or any of the deliveryagents described in U.S. Pat. No. 7,744,910B2, the contents of which areincorporated herein by reference in their entirety), a pharmaceuticallyacceptable buffer, a disintegrant, a detergent,hydroxypropylmethylcellulose, colorants, flavors and sweeteners.

In other embodiments, pharmaceutical compositions comprise one or moreactive polypeptide ingredients together with ethanol, soy phosphatidylcholine, glycerol diolate which is injected into an excess of salinesolution as described in US patent application 2008/0146490A1, thecontents of which are incorporated herein by reference in theirentirety.

The delivery of one or more polypeptides to a subject in need thereofcan be achieved in a number of different ways. In vivo delivery can beperformed directly by administering a composition comprising one or morepolypeptides, to a subject. Alternatively, delivery can be performedindirectly by administering one or more vectors that encode and directthe expression of the polypeptides.

Local delivery avoids gut permeability and systemic exposure. Forexample, polypeptides and/or polypeptide compositions of the presentinvention may be used in the eye as a drop or in the posterior sectionof the eye by direct injection. They may be applied in the gut to targetenzymes. They may be used topically in dermatologic applications (e.g.,creams, ointments, transdermal patches).

Polypeptides and/or polypeptide compositions of the present inventionmay comprise or be formulated with one or more fusogenic agents. As usedherein, the term “fusogenic agent” refers to an agent that is responsiveto changes, such as pH changes in the environment for example. Uponencountering the pH of an endosome, a fusogenic agent can cause aphysical change, e.g., a change in osmotic properties that disrupts orincreases the permeability of the endosome membrane. Preferably, thefusogenic agent changes charge, e.g., becomes protonated, at pH lowerthan physiological range. For example, the fusogenic agent can becomeprotonated at pH 4.5-6.5. A fusogenic agent may serve to release apolypeptide into the cytoplasm of a cell after a composition is takenup, e.g., via endocytosis, by the cell, thereby increasing the cellularconcentration of the polypeptide in the cell.

In some embodiments, fusogenic agents may have a moiety, e.g., an aminogroup, which, when exposed to a specified pH range, will undergo achange, e.g., in charge, e.g., protonation. Changes in charge offusogenic agents can trigger changes, e.g., osmotic changes, invesicles, e.g., endocytic vesicles, e.g., endosomes. For example, thefusogenic agent, upon being exposed to the pH environment of anendosome, will cause a solubility or osmotic change substantial enoughto increase the porosity of (preferably, to rupture) the endosomalmembrane.

Fusogenic agents may be polymers, preferably polyamino chains, e.g.,polyethyleneimine (PEI). PEI may be linear, branched, synthetic ornatural. PEI may be, e.g., alkyl substituted PEI, or lipid substitutedPEI.

In other embodiments, fusogenic agents may be polyhistidine,polyimidazole, polypyridine, polypropyleneimine, mellitin, or polyacetalsubstances, e.g., cationic polyacetals. In some embodiments, fusogenicagents may have an alpha helical structure. Fusogenic agents may bemembrane disruptive agents, e.g., mellittin. Other suitable fusogenicagents can be tested and identified by a skilled artisan.

Polypeptides and/or polypeptide compositions of the present inventionmay comprise or be formulated with one or more condensing agents.Condensing agents of compositions described herein may interact with(e.g., attract, hold, or bind to) polypeptides and act to (a) condense,e.g., reduce the size or charge of polypeptides and/or (b) protectpolypeptides, e.g., protect polypeptides against degradation. Condensingagents may include a moiety, e.g., a charged moiety, which can interactwith polypeptides by ionic interactions. Condensing agents wouldpreferably be charged polymers, e.g., polycationic chains. Condensingagents can be polylysine (PLL), spermine, spermidine, polyamine,pseudopeptide-polyamine, peptidomimetic polyamine, dendrimer polyamine,arginine, amidine, protamine, cationic lipid, cationic porphyrin,quarternary salt of a polyamine, or an alpha helical peptide.

In some embodiments, polypeptides of the present invention may bebicyclic polypeptides. As used herein, the term “bicyclic polypeptide”refers to a polypeptide with two loops. As a non-limiting example,bicyclic polypeptide inhibitors of C5 may be produced in combinatoriallibraries. The bicyclic polypeptides may have 2, 3, 4, 5, 6 or moreamino acids per loop.

In some embodiments, polypeptides and/or polypeptide compositions of thepresent invention may be provided as prodrugs. As used herein, the term“prodrug” refers to a drug that is provided in an inactive form thatbecomes active at some point after administration. In some embodimentswherein polypeptides are administered in the form of a prodrug, aminoacids critical to polypeptide inhibitory activity are unavailable tointeract with the target due to a reversible chemical bond, e.g., anester bond. Upon administration, such prodrugs may be subject tocleavage of the reversible chemical bond, e.g., through enzymatic oracid hydrolysis in the stomach, blood and/or cells of a given targettissue.

C5 Inhibitors

Some polypeptides and/or polypeptide compositions of the presentinvention inhibit complement activation at the level of complementcomponent C5, referred to herein as “C5 inhibitors.” Some C5 inhibitorsfunction by preventing the cleavage of C5 to the cleavage products C5aand C5b, such inhibitors are referred to herein as “C5 cleavageinhibitors.” In some embodiments, methods of the present invention maycomprise inhibiting C5 cleavage in a system. As used herein, a “system”refers to a group of related parts that function together. Such systemsinclude those comprising C5, referred to here as “C5 systems.” C5systems may include, but are not limited to solutions, matrices, cells,tissues, organs, and bodily fluids (including, but not limited toblood.) In some cases, C5 systems may be cellular systems. As usedherein the term “cellular system” refers to a system that comprises oneor more cells or one or more components or products of a cell. In somecases, C5 systems may include in vivo systems, in vitro systems and exvivo systems. In vivo C5 systems may comprise or be comprised in asubject.

In some cases, C5 inhibitors of the invention may include any of thepolypeptides listed in Table 1.

TABLE 1 Compounds of the invention SEQ Compound ID Number Sequence NO.R3000 Ac-Nvl-C-Y-K-N-Y-H-azaTrp-E-Y-P-Tbg-Y-NH2 1 R3001Ac-Nvl-C-Y-E-N-Tbg-Y-azaTrp-E-Y-(N-Me)G-Nvl-(N-Me)S-NH2 2 R3002Ac-Nvl-C-Y-E-N-Tbg-Y-azaTrp-E-Y-P-Phg-Nvl-NH2 3 R3003Ac-Nvl-C-Y-E-N-Tbg-Y-azaTrp-E-Y-P-Phg-P-NH2 4 R3004Ac-Nvl-C-Y-N-N-Tbg-E-azaTrp-E-Y-P-Phg-Tbg-NH2 5 R3005Ac-Nvl-C-Y-azaTrp-(N-Me)G-Tbg-Nvl-azaTrp-E-Y-P-Phg-P-NH2 6 R3006Ac-Y-E-N-Tbg-Y-azaTrp-E-Y-(N-Me)G-Nvl-(N-Me)S-NH2 7 R3007[mXylyl(2,7)]Ac-Nvl-C-K-E-Phg-Y-C-(N-Me)S-Tbg-K-azaTrp-E-Y- 8 NH2 R3008[mXylyl(2,10)]Ac-Nvl-C-Phg-T-azaTrp-E-Y-(N-Me)S-H-C-Nvl-P- 9 Nvl-NH2R3020 [mXylyl(2,7)]M-C-S-E-R-Y-C-E-V-R-W-E-Y-NH2 10 R3021[mXylyl(2,7)]M-C-V-E-R-F-C-D-V-Y-W-E-F-NH2 11 R3079Nvl-Nvl-Y-E-N-Tbg-Y-azaTrp-E-Y-P-Phg-Nvl-NH2 12 R3055Ac-Nvl-S-Y-E-N-Tbg-Y-azaTrp-E-Y-P-Phg-Nvl-NH2 13 R3120Ac-Nvl-Nvl-Y-E-(N-Me)N-Tbg-Y-azaTrp-E-Y-P-Chg-Nvl-NH2 14 R3057[mXylyl(2,7)]M-C-V-E-R-F-C-D-Tbg-Y-azaTrp-E-Y-P-Phg-Nvl- 15 NH2 R3056Ac-Nvl-Nvl-Y-E-N-Tbg-Y-azaTrp-E-Y-P-Phg-Nvl-NH2 16 R3054Ac-Nvl-C-Y-E-N-Tbg-Y-azaTrp-E-Y-P-Chg-Nvl-NH2 17 R3029Ac-Nvl-C-Y-E-N-Tbg-Y-azaTrp-E-Y-P-Phg-NH2 18 R3048[mXylyl(1,6)]Ac-C-V-E-R-F-C-D-V-Y-W-E-F-NH2 19 R3072Ac-Nvl-Nvl-Y-E-N-Tbg-Y-azaTrp-E-Y-P-Chg-Nvl-K-NH2 20 R3024Ac-C-Y-E-N-Tbg-Y-azaTrp-E-Y-P-Phg-Nvl-NH2 21 R3114Ac-Nvl-Nvl-(N-Me)Y-E-N-Tbg-Y-azaTrp-E-Y-P-Chg-Nvl-NH2 22 R3050[pXylyl(2,10)]Ac-Nvl-C-Phg-T-azaTrp-E-Y-(N-Me)S-H-C-Nvl-NH2 23 R3025Ac-Y-E-N-Tbg-Y-azaTrp-E-Y-P-Phg-Nvl-NH2 24 R3061Ac-Nvl-S-Y-E-A-Tbg-Y-azaTrp-E-Y-P-Chg-Nvl-NH2 25 R3041Ac-Y-E-N-Tbg-Y-W-E-Y-P-Phg-Nvl-NH2 26 R3077Ac-Nvl-Nvl-Y-E-N-Tbg-Y-azaTrp-E-Y-P-Chg-Nvl-K-(PEG2000) 27 NH2 R3030Ac-Nvl-C-Y-E-N-Tbg-Y-azaTrp-E-Y-P-NH2 28 R3062Ac-Nvl-S-Y-E-N-A-Y-azaTrp-E-Y-P-Chg-Nvl-NH2 29 R3066Ac-Nvl-S-Y-E-N-Tbg-A-azaTrp-E-Y-P-Chg-Nvl-NH2 30 R3011[mXylyl(2,10)]Ac-Nvl-C-Phg-T-azaTrp-E-Y-(N-Me)S-H-C-Nvl-P- 31 NH2 R3070Ac-Nvl-S-Y-E-N-Tbg-Y-azaTrp-E-Y-A-Chg-Nvl-NH2 32 R3071Ac-Nvl-S-Y-E-N-Tbg-Y-azaTrp-E-Y-P-A-Nvl-NH2 33 R3033[mXylyl(2,10)]Ac-Nvl-C-Phg-A-azaTrp-E-Y-(N-Me)S-H-C-Nvl-NH2 34 R3038[mXylyl(2,10)]Ac-Nvl-C-Phg-T-azaTrp-E-Y-(N-Me)S-A-C-Nvl-NH2 35 R3012[mXylyl(2,10)]Ac-Nvl-C-Phg-T-azaTrp-E-Y-(N-Me)S-H-C-Nvl-NH2 36 R3060Ac-Nvl-S-Y-A-N-Tbg-Y-azaTrp-E-Y-P-Chg-Nvl-NH2 37 R3039[mXylyl(2,10)]Ac-Nvl-C-Phg-T-azaTrp-E-Y-(N-Me)S-H-C-A-NH2 38 R3037[mXylyl(2,10)]Ac-Nvl-C-Phg-T-azaTrp-E-Y-(N-Me)A-H-C-Nvl-NH2 39 R3076Ac-Nvl-Nvl-Y-E-N-Tbg-Y-azaTrp-E-Y-P-Chg-Nvl-K-(BODIPY- 40 TMR-X)NH2R3074 [mXylyl(2,10)]Ac-Nvl-C-Phg-T-azaTrp-E-Tyr(OMe)-(N-Me)S-H-C- 41Nvl-NH2 R3013 [mXylyl(2,10)]Ac-Nvl-C-Phg-T-azaTrp-E-Y-(N-Me)S-H-C-NH2 42R3065 [pXylyl(2,10)]Ac-Nvl-C-Phg-T-azaTrp-E-Y-P-H-C-Nvl-NH2 43 R3073[mXylyl(2,10)]Ac-Nvl-C-Phg-T-azaTrp-E-Phe(4-F)-(N-Me)S-H-C- 44 Nvl-NH2R3116 Ac-Nvl-Nvl-Y-E-N-Tbg-Y-(N-Me)W-E-Y-P-Chg-Nvl-NH2 45 R3091[mXylyl(2,10)]Ac-Nvl-C-Phg-T-W-E-Y-(N-Me)S-A-C-Nvl-NH2 46 R3078PEG2000-Nvl-Nvl-Y-E-N-Tbg-Y-azaTrp-E-Y-P-Phg-Nvl-NH2 47 R3100[mXylyl(2,10)]Ac-Nvl-C-Phg-T-azaTrp-E-F-(N-Me)S-A-C-Nvl-NH2 48 R3121Ac-Nvl-Nvl-Y-E-N-Tbg-Y-azaTrp-E-Y-P-(N-Me)Phg-Nvl-NH2 49 R3043[mXylyl(2,7)]M-C-V-E-R-F-C-D-V-Y-W-E-NH2 50 R3102[mXylyl(2,10)]Ac-Nvl-C-Phg-T-azaTrp-E-Y-P-H-C-Nvl-NH2 51 R3026Ac-E-N-Tbg-Y-azaTrp-E-Y-P-Phg-Nvl-NH2 52 R3031[mXylyl(2,10)]Ac-A-C-Phg-T-azaTrp-E-Y-(N-Me)S-H-C-Nvl-NH2 53 R3019[mXylyl(2,14)]Ac-Nvl-C-Y-E-N-Tbg-Y-azaTrp-E-Y-P-Phg-Nvl-C- 54 NH2 R3014[mXylyl(1,9)]Ac-C-Phg-T-azaTrp-E-Y-(N-Me)S-H-C-Nvl-P-Nvl- 55 NH2 R3104[pXylyl(2,10)]Ac-Nvl-homoC-Phg-T-azaTrp-E-Y-(N-Me)S-H-C-Nvl- 56 NH2R3059 Ac-Nvl-S-A-E-N-Tbg-Y-azaTrp-E-Y-P-Chg-Nvl-NH2 57 R3115Ac-Nvl-Nvl-Y-(N-Me)E-N-Tbg-Y-azaTrp-E-Y-P-Chg-Nvl-NH2 58 R3110Ac-Y-E-N-Tbg-Y-(1-Me)W-E-Y-P-Phg-Nvl-NH2 59 R3126Ac-Nvl-C-Y-N-N-Tbg-E-azaTrp-E-C-P-Phg-Tbg-NH2 60 R3049[oXylyl(2,10)]Ac-Nvl-C-Phg-T-azaTrp-E-Y-(N-Me)S-H-C-Nvl-NH2 61 R3069Ac-Nvl-S-Y-E-N-Tbg-Y-azaTrp-E-A-P-Chg-Nvl-NH2 62 R3015[mXylyl(1,9)]Ac-C-Phg-T-azaTrp-E-Y-(N-Me)S-H-C-NH2 63 R3068Ac-Nvl-S-Y-E-N-Tbg-Y-azaTrp-A-Y-P-Chg-Nvl-NH2 64 R3105[mXylyl(2,10)]Ac-Nvl-C-Phg-T-azaTrp-E-Y-(N-Me)S-H-homoC- 65 Nvl-NH2R3106 [pXylyl(2,10)]Ac-Nvl-C-Phg-T-azaTrp-E-Y-(N-Me)S-H-homoC-Nvl- 66NH2 R3111 [mXylyl(4,10)]Ac-Nvl-T-Phg-C-azaTrp-E-Y-(N-Me)S-A-C-Nvl-NH2 67R3112 [mXylyl(2,10)]Ac-N1e-C-Phg-T-azaTrp-E-Y-(N-Me)S-H-C-Nvl-NH2 68R3113 [mXylyl(3,11)]Ac-Y-Nvl-C-Phg-T-azaTrp-E-Y-(N-Me)S-H-C-Nvl- 69 NH2R3134 [mXylyl(2,10)]Ac-Nvl-C-Phg-T-azaTrp-E-(3-Cl-Phe)-(N-Me)S-A-C- 70Nvl-NH2 R3018[mXylyl(2,10)]Ac-Nvl-C-Y-E-N-Tbg-Y-azaTrp-E-C-P-Phg-Nvl-NH2 71 R3027Ac-N-Tbg-Y-azaTrp-E-Y-P-Phg-Nvl-NH2 72 R3028Ac-Tbg-Y-azaTrp-E-Y-P-Phg-Nvl-NH2 73 R3032[mXylyl(2,10)]Ac-Nvl-C-A-T-azaTrp-E-Y-(N-Me)S-H-C-Nvl-NH2 74 R3058[pXylyl(2,10)]Ac-Nvl-C-Chg-T-azaTrp-E-Y-(N-Me)S-H-C-Nvl-NH2 75 R3067Ac-Nvl-S-Y-E-N-Tbg-Y-A-E-Y-P-Chg-Nvl-NH2 76 R3117Ac-Nvl-Nvl-Y-E-N-Tbg-Y-azaTrp-E-(N-Me)Y-P-Chg-Nvl-NH2 77 R3022Ac-Nvl-C-Phg-T-azaTrp-E-Y-(N-Me)S-H-C-Nvl-P-Nvl-NH2 78 R3016[mXylyl(1,9)]Ac-C-Tbg-Y-azaTrp-E-Y-(N-Me)S-H-C-NH2 79 R3089[mXylyl(2,10)]Ac-Chg-C-Phg-T-azaTrp-E-Y-(N-Me)S-A-C-Nvl-NH2 80 R3083[mXylyl(2,10)]Ac-V-C-Phg-T-azaTrp-E-Y-(N-Me)S-A-C-Nvl-NH2 81 R3087[mXylyl(2,10)]Ac-Nvl-C-(2-OMe)Phg-T-azaTrp-E-Y-(N-Me)S-H-C- 82 Nvl-NH2R3103 [mXylyl(2,10)]Ac-Nvl-homoC-Phg-T-azaTrp-E-Y-(N-Me)S-H-C- 83Nvl-NH2 R3135 [mXylyl(2,10)]Ac-Nvl-C-Phg-T-azaTrp-E-Y-(N-Me)S-(D-Ala)-C-84 Nvl-NH2 R3034 [mXylyl(2,10)]Ac-Nvl-C-Phg-T-A-E-Y-(N-Me)S-H-C-Nvl-NH285 R3035 [mXylyl(2,10)]Ac-Nvl-C-Phg-T-azaTrp-A-Y-(N-Me)S-H-C-Nvl-NH2 86R3036 [mXylyl(2,10)]Ac-Nvl-C-Phg-T-azaTrp-E-A-(N-Me)S-H-C-Nvl-NH2 87R3044 [mXylyl(2,7)]M-C-V-E-R-F-C-D-V-Y-W-NH2 88 R3080[mXylyl(2,9)]Ac-Nvl-C-Phg-T-azaTrp-E-Y-(N-Me)S-C-Nvl-NH2 89 R3085[mXylyl(2,10)]heptanoyl-Nvl-C-Phg-T-azaTrp-E-Y-(N-Me)S-A-C- 90 Nvl-NH2R3086 [mXylyl(5,13)]Ac-Nvl-S-Y-E-C-Tbg-Y-azaTrp-E-Y-P-Chg-C-Nvl- 91 NH2R3092 [mXylyl(2,10)]Ac-Nvl-C-Phg-T-F-E-Y-(N-Me)S-A-C-Nvl-NH2 92 R3095[mXylyl(2,10)]Ac-Nvl-C-Phg-T-azaTrp-E-(homo)F-(N-Me)S-A-C- 93 Nvl-NH2R3096 [mXylyl(2,10)]Ac-Nvl-C-Phg-Aib-azaTrp-E-Y-(N-Me)S-H-C-Nvl- 94 NH2R3122 [mXylyl(2,10)]Ac-Nvl-C-Tiq-T-azaTrp-E-Y-(N-Me)S-H-C-Nvl-NH2 95R3075 [mXylyl(2,11)]Nvl-C-Y-(N-Me)S-Phg-(N-Me-4-F)Phe-(N-Me)S-H-96(N-Me-4-F)Phe-G-C-NH2 R3107[mXylyl(2,10)]Ac-Nvl-homoC-Phg-T-azaTrp-E-Y-(N-Me)S-H- 97 homoC-Nvl-NH2R3108 [pXylyl(2,10)]Ac-Nvl-homoC-Phg-T-azaTrp-E-Y-(N-Me)S-H- 98homoC-Nvl-NH2 R3127[mXylyl(2,10)]Ac-Nvl-C-Y-N-N-Tbg-E-azaTrp-E-C-P-Phg-Tbg-NH2 99 R3133[mXylyl(2,10)]Ac-Nvl-C-Phg-(D-Ala)-azaTrp-E-Y-(N-Me)S-H-C- 100 Nvl-NH2R3009 [mXylyl(2,10)]Ac-Nvl-C-Y-E-(N-Me)G-Tbg-Y-azaTrp-E-C-Nvl-P- 101Nvl-NH2 R3010[mXylyl(2,13)]Ac-Nvl-C-Y-E-(N-Me)G-Tbg-Y-azaTrp-E-Nvl-Nvl-P- 102 C-NH2R3017 [mXylyl(2,8)]Ac-Nvl-C-Y-E-N-Tbg-Y-C-E-Y-P-Phg-Nvl-NH2 103 R3023Ac-Y-P-Y-C-Phg-azaTrp-Tbg-E-Nvl-N-Y-Nvl-E-NH2 104 R3040[cyclo(2,10)]Ac-Nvl-C-Phg-T-azaTrp-E-Y-(N-Me)S-H-C-Nvl-P-Nvl 105 R3042[cyc1o(2,10)]Ac-Nvl-C-Phg-T-azaTrp-E-Y-(N-Me)S-H-C-Nvl-NH2 106 R3045[mXylyl(2,7)]M-C-V-E-R-F-C-D-V-Y-NH2 107 R3046[mXylyl(2,7)]M-C-V-E-R-F-C-D-V-NH2 108 R3047[mXylyl(2,7)]M-C-V-E-R-F-C-NH2 109 R3051[mXylyl(2,11)]Nvl-C-Y-(N-Me)S-Phg-(N-Me-4-F)Phe-(N-Me)S-H- 110(N-Me-4-F)Phe-(N-Me)G-C-NH2 R3052[mXylyl(2,9)]Nvl-C-Y-Tbg-Phg-N-(N-Me)G-L-C-Phg-(N-Me)A- 111 NH2 R3053[mXylyl-bicyclo]Nvl-C-C-N-Tbg-Phg-C-Tbg-(N-Me)S-C-Tbg-NH2 112 R3063Ac-Tbg-Y-azaTrp-E-Y-NH2 113 R3064 Ac-Y-azaTrp-E-Y-P-NH2 114 R3081Ac-Y-E-N-Tbg-Y-azaTrp-(N-Me)E-Y-P-Phg-Nvl-NH2 115 R3082[mXylyl(1,9)]heptanoyl-C-Phg-T-azaTrp-E-Y-(N-Me)S-A-C-Nvl- 116 NH2 R3084[mXylyl(2,10)]Ac-Nvl-C-Phg-T-azaTrp-E-Y-S-A-C-Nvl-NH2 117 R3088[mXylyl(2,10)]Ac-Nvl-C-Phg-T-(5-F)W-E-Y-(N-Me)S-A-C-Nvl- 118 NH2 R3090[mXylyl(2,10)]Ac-Nvl-C-F-T-azaTrp-E-Y-(N-Me)S-A-C-Nvl-NH2 119 R3093[mXylyl(2,10)]Ac-Nvl-C-(D-Chg)-T-azaTrp-E-Y-(N-Me)S-A-C-Nvl- 120 NH2R3094 Ac-Y-E-N-Tbg-Y-(5-MeO)W-E-Y-P-Phg-Nvl-NH2 121 R3097[mXylyl(2,10)]Ac-Nvl-C-Phg-T-azaTrp-D-Y-(N-Me)S-A-C-Nvl-NH2 122 R3098[mXylyl(2,10)]Ac-Nvl-C-Phg-T-azaTrp-Q-Y-(N-Me)S-A-C-Nvl-NH2 123 R3099[mXylyl(2,10)]Ac-Nvl-C-Phg-T-azaTrp-N-Y-(N-Me)S-A-C-Nvl-NH2 124 R3101[mXylyl(2,10)]Ac-Nvl-C-Phg-T-azaTrp-E-Y-(N-Me)S-H-G-C-Nvl- 125 NH2 R3109[mXylyl(2,10)]Ac-Nvl-C-Phg-T-(1-Me-W)-E-Y-(N-Me)S-A-C-Nvl- 126 NH2 R3118Ac-Y-E-N-Tbg-Y-(D-Trp)-E-Y-P-Phg-Nvl-NH2 127 R3119Ac-Y-E-N-Y-(D-Trp)-E-Y-P-Phg-Nvl-NH2 128 R3123Ac-Y-E-N-Tbg-Y-azaTrp-(D-Glu)-Y-P-Phg-Nvl-NH2 129 R3124[mXylyl(1,6)]Ac-C-V-E-R-F-C-V-Y-W-E-F-NH2 130 R3125[mXylyl(1,6)]Ac-C-V-E-R-F-C-W-E-F-NH2 131 R3128Ac-Nvl-C-Y-N-N-Tbg-E-C-E-Y-P-Phg-Tbg-NH2 132 R3129[mXylyl(2,8)]Ac-Nvl-C-Y-N-N-Tbg-E-C-E-Y-P-Phg-Tbg-NH2 133 R3130Ac-Nvl-Nvl-Y-E-N-Tbg-(N-Me)Y-azaTrp-E-Y-P-Chg-Nvl-NH2 134 R3131[mXylyl(2,10)]Ac-Nvl-C-Phg-T-W-Asp(T)-Y-(N-Me)S-H-C-Nvl- 135 NH2 R3132[mXylyl(2,10)]Ac-Nvl-C-Phg-T-(D-Trp)-E-Y-(N-Me)S-H-C-Nvl- 136 NH2 R3136[mXylyl(2,10)]heptanoyl-Nvl-C-(D-Phg)-T-azaTrp-E-Y-(N-Me)S-A- 137C-Nvl-NH2 R3137[mXylyl(1,9)]heptanoyl-C-(D-Phg)-T-azaTrp-E-Y-(N-Me)S-A-C- 138 Nvl-NH2R3138 [mXylyl(1,6)]Ac-C-V-E-R-F-C-D-Tbg-Y-W-E-F-NH2 139 R3139[mXylyl(1,6)]Ac-C-Tbg-E-R-F-C-D-Tbg-Y-W-E-F-NH2 140 R3140[mXylyl(1,6)]Ac-C-V-E-R-F-C-D-V-Y-W-E-Y-P-NH2 141 R3141[mXylyl(1,6)]Ac-C-V-E-R-F-C-D-V-Y-W-E-F-P-NH2 142 R3142[mXylyl(1,6)]Ac-C-V-E-R-F-C-D-V-Y-azaTrp-E-Y-P-NH2 143 R3143[mXylyl(1,6)]Ac-C-V-E-R-F-C-D-Tbg-Y-W-E-Y-P-NH2 144 R3144[mXylyl(1,6)]Ac-C-V-E-R-F-C-D-Tbg-Y-azaTrp-E-Y-P-Phg-Nvl- 145 NH2 R3145[mXylyl(1,6)]Ac-C-V-E-R-F-C-D-Tbg-Y-azaTrp-E-Y-P-(D-Phg)- 146 Nvl-NH2R3146 [mXylyl(1,6)]Ac-C-Tbg-E-R-F-C-D-V-Y-W-E-F-NH2 147 R3147[mXylyl(1,6)]Ac-C-V-E-R-F-C-D-V-Y-W-E-F-Propargyl-Gly-NH2 148 R3148[mXylyl(1,6)]Ac-C-A-E-R-F-C-D-Tbg-Y-W-E-Y-P-Phg-Nvl-NH2 149 R3149[mXylyl(1,6)]Ac-C-A-E-R-F-C-D-Tbg-Y-W-E-Y-P-(D-Phg)-Nvl- 150 NH2 R3150[mXylyl(1,6)]Ac-C-V-A-R-F-C-D-Tbg-Y-azaTrp-E-Y-P-Phg-Nvl- 151 NH2 R3151[mXylyl(1,6)]Ac-C-V-A-R-F-C-D-Tbg-Y-azaTrp-E-Y-P-(D-Phg)- 152 Nvl-NH2R3152 [mXylyl(1,6)]Ac-C-V-E-R-F-C-D-Tbg-Y-azaTrp-E-Y-P-Chg-Nvl- 153 NH2R3153 [mXylyl(1,6)]Ac-C-V-E-A-F-C-D-Tbg-Y-azaTrp-E-Y-P-Phg-Nvl- 154 NH2R3154 [mXylyl(1,6)]Ac-C-V-E-A-F-C-D-Tbg-Y-azaTrp-E-Y-P-(D-Phg)- 155Nvl-NH2 R3155 [mXylyl(1,6)]Ac-C-V-E-R-A-C-D-Tbg-Y-azaTrp-E-Y-P-Phg-Nvl-156 NH2 R3156 [mXylyl(1,6)]Ac-C-V-E-R-A-C-D-Tbg-Y-azaTrp-E-Y-P-(D-Phg)-157 Nvl-NH2 R3157[mXylyl(1,6)]Ac-C-V-E-R-F-C-A-Tbg-Y-azaTrp-E-Y-P-Phg-Nvl- 158 NH2 R3158[mXylyl(1,6)]Ac-C-V-E-R-F-C-A-Tbg-Y-azaTrp-E-Y-P-(D-Phg)- 159 Nvl-NH2R3159 [mXylyl(1,6)](des-amino)C-V-E-R-F-C-D-Tbg-Y-azaTrp-E-Y-P-Phg- 160Nvl-NH2 R3160[mXylyl(1,6)](des-amino)C-V-E-R-F-C-D-Tbg-Y-azaTrp-E-Y-P-(D- 161Phg)-Nvl-NH2 R3161[mXylyl(1,6)]Ac-C-A-E-R-F-C-D-Tbg-Y-azaTrp-E-Y-P-Phg-K-NH2 162 R3162[mXylyl(1,6)]Ac-C-A-E-R-F-C-D-Tbg-Y-azaTrp-E-Y-P-(D-Phg)-K- 163 NH2R3163 [cyclo(1,6)]Ac-C-V-E-R-F-C-D-Tbg-Y-azaTrp-E-Y-P-Phg-Nvl-NH2 164R3164 [mXylyl(1,6)]Ac-C-A-E-R-F-C-D-Tbg-Y-azaTrp-E-Y-P-Phg-(Lys- 165C12)-NH2 R3165[mXylyl(1,6)]Ac-C-A-E-R-F-C-D-Tbg-Y-azaTrp-E-Y-P-Phg-(Lys- 166 C10)-NH2R3166 [mXylyl(1,6)]Ac-C-A-E-R-F-C-D-Tbg-Y-azaTrp-E-Y-P-Phg-(Lys- 167C8)-NH2 R3167[mXylyl(1,6)]Ac-C-V-E-R-F-C-(alpha-methyl)D-Tbg-Y-azaTrp-E-Y- 168P-Chg-Nvl-NH2 R3168[mXylyl(1,6)]Ac-C-V-E-R-F-C-Asp(T)-Tbg-Y-azaTrp-E-Y-P-Chg- 169 Nvl-NH2R3169 [cyclo(1,6)]Ac-K-V-E-R-F-D-D-Tbg-Y-azaTrp-E-Y-P-Chg-Nvl-NH2 170R3170 [mXylyl(1,6)]Ac-C-A-E-R-F-C-D-Tbg-Y-azaTrp-E-Y-P-Phg-K 171 R3171[mXylyl(1,6)]Ac-C-A-E-R-F-C-D-Tbg-Y-azaTrp-E-Y-P-Phg-(Lys- 172 C12)R3172 [mXylyl(1,6)]Ac-C-V-E-R-F-C-(N-Me)D-Tbg-Y-azaTrp-E-Y-P-Chg- 173Nvl-NH2 R3173 [cyclo(1,6)]Ac-K-V-E-R-F-D-D-Tbg-Y-azaTrp-E-Y-P-Chg-Nvl174 R3174 [cyclo(1,6)]Ac-K-V-E-R-F-D-Asp(T)-Tbg-Y-azaTrp-E-Y-P-Chg-Nvl175 R3175 [cyclo(1,6)]Ac-K-V-E-R-F-D-D-Tbg-Y-azaTrp-E-Y-P-Chg-B20 176R3176 [cyclo(1,6)]Ac-K-V-E-R-F-D-(N-Me)D-Tbg-Y-azaTrp-E-Y-P-Chg- 177 NvlR3177 [mXylyl(1,6)]Ac-C-V-E-R-F-C-D-Tbg-Y-azaTrp-E-W-P-Chg-Nvl 178 R3178[mXylyl(1,6)]Ac-C-V-E-R-F-C-D-Tbg-Y-azaTrp-E-(homo)Phe-P- 179 Chg-NvlR3179 [mXylyl(1,6)]Ac-C-V-E-R-F-C-D-Tbg-Y-azaTrp-E-(m-Cl-homo)Phe- 180P-Chg-Nvl R3180[mXylyl(1,6)]Ac-C-V-E-R-F-C-D-Tbg-Y-azaTrp-E-2Nal-P-Chg-Nvl 181 R3181[mXylyl(1,6)]Ac-C-V-E-R-F-C-D-Tbg-Y-(3-aminomethyl)Phe-E-Y- 182P-Chg-Nvl R3182[cyclo-triazolyl(1,6)]Ac-X02-V-E-R-F-X31-D-Tbg-Y-azaTrp-E-Y-P- 183Chg-Nvl R3183 [cyclo(1,6)]Ac-K-V-E-R-F-D-(N-Me)D-Tbg-Y-azaTrp-E-Y-P-Chg-184 (Lys-C16) R3184[cyclo-thioalkyl(1,5)]V-E-R-F-C-D-Tbg-Y-azaTrp-E-Y-P-Chg-Nvl 185 R3185[mXylyl(1,6)]Ac-C-V-E-R-F-C-Cle-Tbg-Y-azaTrp-E-Y-P-Chg-Nvl 186 R3186[mXylyl(1,6)]Ac-C-V-E-R-F-C-(Ac-Pyran)-Tbg-Y-azaTrp-E-Y-P- 187 Chg-NvlR3187 [mXylyl(1,6)]Ac-C-V-E-R-F-C-D-Tbg-Y-azaTrp-E-(3- 188aminomethyl)Phe-P-Chg-Nvl R3188[cyclo-olefinyl(1,6)]Ac-X30-V-E-R-F-X12-D-Tbg-Y-azaTrp-E-Y-P- 189Chg-Nvl R3189 [mXylyl(1,6)]Ac-C-A-E-R-F-C-D-Tbg-Y-azaTrp-E-Y-P-Phg-(Lys-190 C16) R3190[cyclo(1,6)]Ac-K-V-E-R-F-D-(N-Me)D-Tbg-Y-azaTrp-E-Y-P-Chg- 191 B20 R3191[cyclo(1,6)]Ac-K-V-E-R-F-D-(N-Me)D-Tbg-Y-azaTrp-E-Y-P-Chg-K 192 R3192[cyclo(1,6)]Ac-K-V-E-R-F-D-(N-Me)D-Tbg-Y-azaTrp-E-Y-P-Chg- 193 K-NH2R3193 [cyclo(1,6)]Ac-K-V-E-R-F-D-(N-Me)D-Tbg-Y-azaTrp-E-Y-P-Chg- 194 B28R3194 [cyclo(1,6)]Ac-K-V-E-R-F-D-(N-Me)D-Tbg-Y-azaTrp-E-Y-P-Chg- 195(Lys-C16)-NH2 R3195[cyclo(1,6)]Ac-K-V-E-R-F-D-(N-Me)D-Tbg-Y-W-E-Y-P-Chg-(Lys- 196 C16)R3196 [cyclo(1,6)]Ac-K-V-E-R-F-D-(N-Me)D-Tbg-Y-W-E-Y-P-Chg-K 197 R3197[cyc1o(1,6)]Ac-K-V-E-R-F-D-(N-Me)D-Tbg-Y-W-E-Y-P-Chg-K14 198 R3198[cyclo(1,6)](desamino)C-V-E-R-F-C-(N-Me)D-Tbg-Y-azaTrp-E-Y-P- 199Chg-(Lys-C16) R3199[cyclo(1,6)](desamino)C-(D-Ala)-E-R-F-C-(N-Me)D-Tbg-Y-azaTrp- 200E-Y-P-Chg-(Lys-C16) R3200[cyclo(1,6)]Ac-K-V-E-R-F-D-(N-Me)D-Tbg-Y-azaTrp-E-Y-P-Aib- 201 (Lys-C16)R3201 [mXylyl(1,6)]Ac-C-V-E-R-F-C-D-Tbg-Y-azaTrp-E-Y-P-Chg-Nvl 211

In C5 systems, C5 and other system components may be in solution or maybe fixed, such as in an assay well. C5 systems may further compriseother components of complement, in some cases including all of thecomponents necessary to form the membrane attack complex (MAC.) In somecases, polypeptides and/or polypeptide compositions of the invention maybe used to inhibit C5 cleavage in a human subject. Such polypeptidesand/or polypeptide compositions may find utility in treating variouscomplement-related disorders and/or diseases as well as accompanyinginflammatory conditions. Certain C5 inhibitors are known in the art andare taught in U.S. Pat. Nos. 7,348,401 and 6,355,245; both of which areherein incorporated by reference in their entireties.

Cleavage of C5 yields the proteolytic products C5a and C5b. The cleavagesite of C5 that is cleaved to yield these products is referred to hereinas the C5a-C5b cleavage site. C5b contributes to the formation of themembrane attack complex (MAC) while C5a stimulates the immune system andthe inflammatory response. In some embodiments, polypeptides and/orpolypeptide compositions of the present invention prevent the cleavageof C5 and therefore may be useful in the treatment of inflammationthrough the inhibition of inflammatory events including, but not limitedto chemotaxis and activation of inflammatory cells (e.g. macrophages,mast cells, neutrophils and platelets), proliferation of endothelialcells and edema.

Many of the components of the complement system, including but notlimited to C3, C4, and C5, are functionally inert in their native stateuntil targeted for cleavage into multiple active components. Cleavage ofC3 or C4 causes a conformational change that exposes an internalthioester domain. Within the domain, an internal thioester linkagebetween cysteine and glutamine residue side chains is a chemicallylabile bond that confers the ability of C3 and C4 to bind cell surfaceand/or biological molecules. The cleavage of C3 and C4 also provides thecomponents of the C5 convertase, either C3bC4bC2a or (C3b)2Bb. (Law, S.K., et al. (1997). Protein Science. 6:263-274; van den Elsen, J. M. H.,(2002). J. Mol. Biol. 322:1103-1115; the contents of each of which areherein incorporated by reference in their entireties.)

The multiple domain structure of C5 is similar to C3 and C4. The C5convertase cleaves C5 into the components C5a and C5b. The cleavage ofC5 causes a conformational change that exposes the C5b thioester-likedomain, which plays a role in C5 binding C6, followed by interactionswith C7 and C8 to form the cytolytic MAC. The domain structures of C5comprise regulatory features that are critical for the processing anddownstream activity of complement. (Fredslund, F. et al. (2008). Nature.9:753-760; Hadders, M. A. et al. (2012). Cell Reports. 1:200-207.)

In some embodiments, compounds of the present invention may bind C5 andprevent cleavage of C5 into C5a and C5b cleavage products.

Recently, a new paradigm for complement activation was proposed, basedupon the discovery that thrombin generates previously unidentified C5products that support the terminal complement activation pathway(Krisinger, et al., (2014). Blood. 120(8):1717-1725).

Thrombin acts in the coagulation cascade, a second circulation-basedprocess by which organisms, in response to injury, are able to limitbleeding, restore vascular integrity, and promote healing. Subsequent tovessel damage, tissue factor is exposed to the circulation, setting offa cascade of proteolytic reactions that leads to the generation of thecentral coagulation enzyme thrombin, which converts fibrinogen into afibrin clot.

Historically, the complement activation pathway has been viewedseparately from the coagulation cascade; however, the interplay of thesetwo systems is worthy of renewed consideration. Coagulation andcomplement are coordinately activated in an overlapping spatiotemporalmanner in response to common pathophysiologic stimuli to maintainhomeostasis, and disease emerges when there is unchecked activation ofthe innate immune and coagulation responses, as evidenced by, forexample, atherosclerosis, stroke, coronary heart disease, diabetes,ischemia-reperfusion injury, trauma, paroxysmal nocturnalhemoglobinuria, age-related macular degeneration, and atypicalhemolytic-uremic syndrome. In fact, introduction of complementinhibitors has been found to simultaneously treat the inflammatory andthrombotic disturbances associated with some of these disorders.

As noted above, the complement system is activated via three mainpathways, all converging with proteolytic activation of the centralcomplement component C3. Subsequently, the formation of C5 convertasesresults in cleavage of C5 at arginine 751 (R751) to liberate achemotactic and anaphylatoxic C5a fragment and generate C5b. C5b is theinitiating factor for assembly of the C5b dependent lytic membraneattack complex (MAC; also known as C5b-9), responsible for destroyingdamaged cells and pathogens.

Several molecular links between complement and coagulation have beenidentified. Most notably in what was described as a new complementactivation pathway, thrombin was found to be capable of directlypromoting activation of complement by cleaving C5, presumably at R751,thereby releasing C5a in the absence of C3 (Huber-Lang, et al., 2006.Nature Med. 12(6):682-687). However, these studies did not comparethrombin with the bona fide C5 convertase, and only limited biochemicalanalyses were performed; thus, the physiologic relevance of the pathwaywas not evaluable.

Using purified and plasma-based systems, the effects of thrombin and C5convertase on C5 were assessed by measuring release of the anaphylatoxinC5a and generation of the C5b, component of MAC. It was discovered that,while thrombin cleaved C5 poorly at R751, yielding minimal C5a and C5b,it efficiently cleaved C5 at a newly identified, highly conserved R947site, generating previously undescribed intermediates C5_(T) andC5b_(T). Tissue factor-induced clotting of plasma led to proteolysis ofC5 at a thrombin-sensitive site corresponding to this new R947 site andnot R751. Combined treatment of C5 with thrombin and C5 convertaseyielded C5a and C5b_(T), the latter forming a C5b_(T)-9 membrane attackcomplex with significantly more lytic activity than with C5b-9. Thus, anew paradigm has been proposed for complement activation, in whichthrombin is an invariant and critical partner with C5 convertase ininitiating formation of a more active MAC via formation of previouslyunidentified C5 products that are generated via cooperative proteolysisby the two enzymes. These discoveries provide new insights into theregulation of innate immunity in the context of coagulation activationoccurring in many diseases. (Krisinger, et al., (2014). Blood.120(8):1717-1725).

In some embodiments, polypeptides and/or polypeptide compositions of theinvention may inhibit thrombin-induced complement activation. Suchpolypeptides and/or polypeptide compositions may therefore be used totreat hemolysis resulting from thrombin-induced complement activation.

Given the findings of molecular links between the complement andcoagulation pathways, it is believed that complement may be activated byadditional components of the coagulation and/or inflammation cascades.For example, other serine proteases with slightly different substratespecificity may act in a similar way. Huber-Lang et al. (2006) showedthat thrombin not only cleaved C5 but also in vitro-generated C3a whenincubated with native C3 (Huber-Lang, et al., 2006. Nature Med.12(6):682-687; the contents of which are herein incorporated byreference in their entirety). Similarly, other components of thecoagulation pathway, such as FXa, FXIa and plasmin, have been found tocleave both C5 and C3.

Specifically, in a mechanism similar to the one observed via thrombinactivation, it has been observed that plasmin, FXa, FIXa and FXIa areable to cleave C5 to generate C5a and C5b (Amara, et al., (2010). J.Immunol. 185:5628-5636; Amara, et al., (2008) “Interaction Between theCoagulation and Complement System” in Current Topics in Complement II,J. D. Lambris (ed.), pp. 71-79). The anaphylatoxins produced were foundto be biologically active as shown by a dose-dependent chemotacticresponse of neutrophils and HMC-1 cells, respectively. Plasmin-inducedcleavage activity could be dose-dependently blocked by the serineprotease inhibitor aprotinin and leupeptine. These findings suggest thatvarious serine proteases belonging to the coagulation system are able toactivate the complement cascade independently of the establishedpathways. Moreover, functional C5a and C3a are generated (as detected byimmunoblotting and ELISA), both of which are known to be cruciallyinvolved in the inflammatory response.

In some embodiments, polypeptides and/or polypeptides compositions ofthe invention may inhibit activation of C5 by plasmin, FXa, FIXa, FXIaand other proteases of the coagulation pathway.

Human leukocyte elastase (HLE), an enzyme secreted by neutrophils andmacrophages during inflammatory processes, has long been known to alsorelease from C5 a chemotactic, C5a-like fragment. However, this C5a-likefragment, is not identical with C5a, as HLE does not cleave peptidebonds at the cleavage site that ordinarily cleaves C5 into C5a and C5bafter the exposure to the complement convertases. Rather, cleavage ofcomplement C5 by HLE has also been found to generate a functionallyactive C5b-like molecule that is able to participate in MAC formation(Vogt, (1999). Immunobiology. 201:470-477).

In some embodiments, polypeptides and/or polypeptides compositions ofthe invention may inhibit activation of C5 by HLE and other proteases ofthe inflammation cascade.

In some embodiments, polypeptides and/or polypeptide compositions of thepresent invention may be useful in the treatment of diseases, disordersand/or conditions where C5 cleavage leads to progression of the disease,disorder and/or condition. Such diseases, disorders and/or conditionsmay include, but are not limited to immune and autoimmune, neurological,cardiovascular, pulmonary, and ocular diseases, disorders and/orconditions. Immune and autoimmune diseases and/or disorders may include,but are not limited to Acute Disseminated Encephalomyelitis (ADEM),Acute necrotizing hemorrhagic leukoencephalitis, Addison's disease,Agammaglobulinemia, Alopecia areata, Amyloidosis, Ankylosingspondylitis, Acute antibody-mediated rejection following organtransplantation, Anti-GBM/Anti-TBM nephritis, Antiphospholipid syndrome(APS), Autoimmune angioedema, Autoimmune aplastic anemia, Autoimmunedysautonomia, Autoimmune hepatitis, Autoimmune hyperlipidemia,Autoimmune immunodeficiency, Autoimmune inner ear disease (AIED),Autoimmune myocarditis, Autoimmune pancreatitis, Autoimmune retinopathy,Autoimmune thrombocytopenic purpura (ATP), Autoimmune thyroid disease,Autoimmune urticaria, Axonal & neuronal neuropathies, Bacterial sepsisand septic shock, Balo disease, Behcet's disease, Bullous pemphigoid,Cardiomyopathy, Castleman disease, Celiac disease, Chagas disease,Chronic fatigue syndrome, Chronic inflammatory demyelinatingpolyneuropathy (CIDP), Chronic recurrent multifocal osteomyelitis(CRMO), Churg-Strauss syndrome, Cicatricial pemphigoid/benign mucosalpemphigoid, Crohn's disease, Cogans syndrome, Cold agglutinin disease,Congenital heart block, Coxsackie myocarditis, CREST disease, Essentialmixed cryoglobulinemia, Demyelinating neuropathies, Dermatitisherpetiformis, Dermatomyositis, Devic's disease (neuromyelitis optica),Diabetes Type I, Discoid lupus, Dressler's syndrome, Endometriosis,Eosinophilic esophagitis, Eosinophilic fasciitis, Erythema nodosum,Experimental allergic encephalomyelitis, Evans syndrome, Fibromyalgia,Fibrosing alveolitis, Giant cell arteritis (temporal arteritis),Glomerulonephritis, Goodpasture's syndrome, Granulomatosis withPolyangiitis (GPA) see Wegener's, Graves' disease, Guillain-Barresyndrome, Hashimoto's encephalitis, Hashimoto's thyroiditis, Hemolyticanemia (including atypical hemolytic uremic syndrome and plasmatherapy-resistant atypical hemolytic-uremic syndrome), Henoch-Schonleinpurpura, Herpes gestationis, Hypogammaglobulinemia, Idiopathicthrombocytopenic purpura (ITP), IgA nephropathy, IgG4-related sclerosingdisease, Immunoregulatory lipoproteins, Inclusion body myositis,Insulin-dependent diabetes (type1), Interstitial cystitis, Juvenilearthritis, Juvenile diabetes, Kawasaki syndrome, Lambert-Eaton syndrome,Large vessel vasculopathy, Leukocytoclastic vasculitis, Lichen planus,Lichen sclerosus, Ligneous conjunctivitis, Linear IgA disease (LAD),Lupus (SLE), Lyme disease, Meniere's disease, Microscopic polyangiitis,Mixed connective tissue disease (MCTD), Mooren's ulcer, Mucha-Habermanndisease, Multiple endocrine neoplasia syndromes, Multiple sclerosis,Multifocal motor neuropathy, Myositis, Myasthenia gravis, Narcolepsy,Neuromyelitis optica (Devic's), Neutropenia, Ocular cicatricialpemphigoid, Optic neuritis, Osteoarthritis, Palindromic rheumatism,PANDAS (Pediatric Autoimmune Neuropsychiatric Disorders Associated withStreptococcus), Paraneoplastic cerebellar degeneration, Paroxysmalnocturnal hemoglobinuria (PNH), Parry Romberg syndrome,Parsonnage-Turner syndrome, Pars planitis (peripheral uveitis),Pemphigus, Peripheral neuropathy, Perivenous encephalomyelitis,Pernicious anemia, POEMS syndrome, Polyarteritis nodosa, Type I, II, &III autoimmune polyglandular syndromes, Polyendocrinopathies,Polymyalgia rheumatica, Polymyositis, Postmyocardial infarctionsyndrome, Postpericardiotomy syndrome, Progesterone dermatitis, Primarybiliary cirrhosis, Primary sclerosing cholangitis, Psoriasis, Psoriaticarthritis, Idiopathic Pulmonary fibrosis, Pyoderma gangrenosum, Pure redcell aplasia, Raynauds phenomenon, Reactive arthritis, Reflexsympathetic dystrophy, Reiter's syndrome, Relapsing polychondritis,Restless legs syndrome, Retroperitoneal fibrosis, Rheumatic fever,Rheumatoid arthritis, Sarcoidosis, Schmidt syndrome, Scleritis,Scleroderma, Shiga-Toxin producing Escherichia Coli Hemolytic-UremicSyndrome (STEC-HUS), Sjogren's syndrome, Small vessel vasculopathy,Sperm & testicular autoimmunity, Stiff person syndrome, Subacutebacterial endocarditis (SBE), Susac's syndrome, Sympathetic ophthalmia,Takayasu's arteritis, Temporal arteritis/Giant cell arteritis,Thrombocytopenic purpura (TTP), Tolosa-Hunt syndrome, Transversemyelitis, Tubular autoimmune disorder, Ulcerative colitis,Undifferentiated connective tissue disease (UCTD), Uveitis,Vesiculobullous dermatosis, Vasculitis, Vitiligo and Wegener'sgranulomatosis (also known as Granulomatosis with Polyangiitis (GPA)).Neurological diseases, disorders and/or conditions may include, but arenot limited to Alzheimer's disease, Parkinson's disease, Lewy bodydementia and Multiple sclerosis. Cardiovascular diseases, disordersand/or conditions may include, but are not limited to atherosclerosis,myocardial infarction, stroke, vasculitis, trauma and conditions arisingfrom cardiovascular intervention (including, but not limited to cardiacbypass surgery, arterial grafting and angioplasty). Pulmonary diseases,disorders and/or conditions may include, but are not limited to asthma,pulmonary fibrosis, chronic obstructive pulmonary disease (COPD) andadult respiratory distress syndrome. Ocular related applicationsinclude, but are not limited to: Age-related macular degeneration,allergic and giant papillary conjunctivitis, Behcet's disease, choroidalinflammation, complications related to intraocular surgery, cornealtransplant rejection, corneal ulcers, cytomegalovirus retinitis, dry eyesyndrome, endophthalmitis, Fuch's disease, Glaucoma, immune complexvasculitis, inflammatory conjunctivitis, ischemic retinal disease,keratitis, macular edema, ocular parasitic infestation/migration,retinitis pigmentosa, scleritis, Stargardt disease, subretinal fibrosis,uveitis, vitreo-retinal inflammation, and Vogt-Koyanagi-Harada disease.

Polypeptides and/or polypeptide compositions of the present inventionmay be particularly useful in the treatment of patients with PNH thatshow a poor response to monoclonal antibody therapies, such asECULIZUMAB® therapy, due to mutations in the C5 gene that preventbinding of the antibody to C5 (Nishimura, J-I. (2012). 54^(th) ASHAnnual Meeting, Abstract 3197).

Polypeptides and/or polypeptide compositions of the present inventionmay be useful in the treatment of infectious diseases, disorders and/orconditions, for example, in a subject having an infection. In somepreferred embodiments the subject has an infection and is at risk ofdeveloping sepsis or a septic syndrome. Polypeptides and/or polypeptidecompositions of the present invention are particularly useful in thetreatment of sepsis.

Polypeptides and/or polypeptide compositions of the present inventionmay also be administered to improve the outcome of clinical procedureswherein complement inhibition is desired. Such procedures may include,but are not limited to grafting, transplantation, implantation,catheterization, intubation and the like. In some embodiments,polypeptides and/or polypeptide compositions of the invention are usedto coat devices, materials and/or biomaterials used in such procedures.In some embodiments, the inner surface of a tube may be coated withpolypeptides and/or polypeptide compositions to prevent complementactivation within a bodily fluid that passes through the tube, either invivo or ex vivo, e.g., extracorporeal shunting, e.g., dialysis andcardiac bypass.

In some embodiments, polypeptides of the invention bind to C5 with 1:1stoichiometry. In some cases, polypeptides of the invention inhibitthrough an allosteric mechanism or by blocking binding of convertase.

In some embodiments, polypeptides of the invention inhibit C5 cleavagein PNH patients with ECULIZUMAB®-resistant C5 polymorphisms.

II. Methods of Use

Included herein are methods of using compounds (e.g., any of thecompounds listed in Table 1) or compositions of the invention to reduceC5 cleavage and downstream consequences including, but not limited to,complement activation, cell lysis, red blood cell lysis (also referredto herein as “hemolysis”). In some cases, such methods may includereducing cleavage of C5 in vitro or in vivo. In some cases, such methodsmay be carried out in a biological system, in an assay, or in a subject.

C5 cleavage, complement activation or hemolysis, according to themethods of the invention, may be reduced from about 1% to about 5%, fromabout 2% to about 10%, from about 5% to about 20%, from about 10% toabout 30%, from about 20% to about 50%, from about 25% to about 75%,from about 30% to about 60%, from about 50% to about 75%, from about 50%to about 90%, from about 50% to about 95%, from about 75% to about 100%,from about 80% to about 100%, from about 85% to about 95%, or from about90% to about 100%. Such reductions may be assessed by examining thelevels of one or more C5 cleavage products (e.g., C5a or C5b proteins).In such embodiments, cleavage product levels may be compared to levelsin an untreated control sample, subject or system or backgroundmeasurement and the percent difference may be determined. Such cleavageproducts may be measured, in some cases, by immunoassay (e.g., EIA orELISA). Similarly, events downstream of C5 cleavage may be measured andcompared to an untreated control sample, subject or system or backgroundmeasurement. In some cases, such events may include the formation of themembrane attack complex (MAC). MAC formation may be measured by ELISA(e.g., WIESLAB® ELISA, Euro Diagnostica, Malmo, Sweden). In some cases,hemolysis may be measured spectrophotochemically (e.g., observingoptical density at about 412 nm wavelength) to detect release ofhemoglobin from ruptured blood cells.

In some embodiments, methods of the invention include methods ofreducing C5 cleavage in a biological system comprising providing apolypeptide of the invention. In some cases, such reduction is assessedby comparison to an untreated control system. Reduction in C5 cleavagemay include reductions of at least 1%, at least 5%, at least 10%, atleast 20%, at least 50%, at least 75%, at least 80%, at least 85%, atleast 90%, at least 95%, at least 98%, at least 99%, or 100%. Accordingto such methods, polypeptides may be provided at concentrations of fromabout 0.001 nM to about 5 nM, from about 0.01 nM to about 10 nM, fromabout 0.1 nM to about 50 nM, from about 1 nM to about 100 nM, from about20 nM to about 200 nM, or from about 100 nM to about 10,000 nM.

In some embodiments, methods of the invention include methods ofreducing hemolysis in a biological system comprising providing apolypeptide of the invention. In some cases, such reduction is assessedby comparison to an untreated control system. Reduction in hemolysis mayinclude reductions of at least 1%, at least 5%, at least 10%, at least20%, at least 50%, at least 75%, at least 80%, at least 85%, at least90%, at least 95%, at least 98%, at least 99%, or 100%. According tosuch methods, polypeptides may be provided at concentrations of fromabout 0.001 nM to about 5 nM, from about 0.01 nM to about 10 nM, fromabout 0.1 nM to about 50 nM, from about 1 nM to about 100 nM, from about20 nM to about 200 nM, or from about 100 nM to about 10,000 nM.

In some embodiments, methods of the invention include methods ofreducing hemolysis in a subject relative to hemolysis levels previouslyobserved in the subject. Reduction in hemolysis may include reductionsof at least 1%, at least 5%, at least 10%, at least 20%, at least 50%,at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, atleast 98%, at least 99%, or 100%. In some cases, reductions in hemolysismay include reductions of from about 1% to about 10%, from about 5% toabout 25%, from about 20% to about 60%, from about 40% to about 80%,from about 50% to about 95%, or from about 60% to 100%. According tosuch methods, polypeptides may be provided at concentrations thatinclude, but are not limited to from about 0.001 nM to about 5 nM, fromabout 0.01 nM to about 10 nM, from about 0.1 nM to about 50 nM, fromabout 1 nM to about 100 nM, from about 20 nM to about 200 nM, or fromabout 100 nM to about 10,000 nM. In some cases, polypeptides may beadministered to human subjects using doses that include, but are notlimited to from about 0.001 mg/kg to about 1 mg/kg, from about 0.01mg/kg to about 2 mg/kg, from about 0.1 mg/kg to about 10 mg/kg, fromabout 1 mg/kg to about 20 mg/kg, or from about 5 mg/kg to about 50mg/kg. In some cases, polypeptide concentrations used for administrationmay be varied to achieve a desired level of the polypeptide in theplasma of the subject receiving the polypeptide. In some cases, desiredplasma levels of polypeptides of the invention may include, but are notlimited to from about 0.001 μM to about 2 μM, from about 0.01 μM toabout 3 μM, from about 0.1 μM to about 20 μM, or from about 1 μM toabout 50 μM.

Therapeutic Indications

The invention relates in particular to the use of polypeptide (e.g.peptidomimetics and cyclic polypeptides) and compositions containing atleast one polypeptide, for the treatment of a disorder, condition ordisease. In some cases, compounds and compositions of the invention maybe used to treat subjects suffering from paroxysmal nocturnalhemoglobinuria (PNH). Subjects with PNH are unable to synthesizefunctional versions of the complement regulatory proteins CD55 and CD59on hematopoietic stem cells. This results in complement-mediatedhemolysis and a variety of downstream complications. Othercomplement-related disorders and diseases include, but are not limitedto autoimmune diseases and disorders, neurological diseases anddisorders, blood diseases and disorders and infectious diseases anddisorders. Experimental evidence suggests that many complement-relateddisorders are alleviated through inhibition of complement activity.

Current treatments for PNH include the use of ECULIZUMAB® (AlexionPharmaceuticals, Cheshire, CT). In some cases, ECULIZUMAB® may beineffective due to mutation in C5, short half-life, immune reaction, orother reason. In some embodiments, methods of the invention includemethods of treating subjects with PNH, wherein such subjects have beentreated previously with ECULIZUMAB®. In some cases, ECULIZUMAB® isineffective in such patients, making treatment with polypeptides of theinvention important for therapeutic relief. In some cases, polypeptidesof the invention are administered simultaneously or in conjunction withECULIZUMAB® therapy. In such cases, such subjects may experience one ormore beneficial effects of such combined treatment, including, but notlimited to more effective relief, faster relief or fewer side effects.

An acquired mutation in the phosphatidylinositol glycan anchorbiosynthesis, class A (PIG-A) gene that originates from a multipotenthematopoietic stem cell results in a rare disease known as paroxysmalnocturnal hemoglobinuria (PNH) (Pu, J. J. et al., Paroxysmal nocturnalhemoglobinuria from bench to bedside. Clin Transl Sci. 2011 June;4(3):219-24). PNH is characterized by bone marrow disorder, hemolyticanemia and thrombosis. The PIG-A gene product is necessary for theproduction of a glycolipid anchor, glycosylphosphatidylinositol (GPI),utilized to tether proteins to the plasma membrane. Twocomplement-regulatory proteins, CD55 and CD59, become nonfunctional inthe absence of GPI. This leads to complement-mediated destruction ofthese cells. Polypeptides and/or polypeptide compositions of the presentinvention are particularly useful in the treatment of PNH.

In some embodiments, methods of the invention include methods ofreducing hemolysis in subjects with PNH. Such methods may includeadministering polypeptides of the invention to such subjects. Accordingto such methods, polypeptides may be administered at doses that include,but are not limited to from about 0.001 mg/kg to about 1 mg/kg, fromabout 0.01 mg/kg to about 2 mg/kg, from about 0.1 mg/kg to about 10mg/kg, from about 1 mg/kg to about 20 mg/kg, or from about 5 mg/kg toabout 50 mg/kg. Additional methods may include single or multiple doses.In cases where multiple doses are administered, administration mayinclude, but is not limited to daily, weekly, monthly, or yearlyadministration.

As used herein the terms “treat,” “treatment,” and the like, refer torelief from or alleviation of pathological processes. In the context ofthe present invention insofar as it relates to any of the otherconditions recited herein below, the terms “treat,” “treatment,” and thelike mean to relieve or alleviate at least one symptom associated withsuch condition, or to slow or reverse the progression or anticipatedprogression of such condition, such as slowing the progression of amalignancy or cancer, or increasing the clearance of an infectiousorganism to alleviate/reduce the symptoms caused by the infection, e.g.,hepatitis caused by infection with a hepatitis virus or reducing thedestruction of red blood cells (as measured by reduced transfusionrequirements or increased hematocrit or hemoglobin levels) resultingfrom paroxysmal nocturnal hemoglobinuria.

By “lower” or “reduce” in the context of a disease marker or symptom ismeant a statistically significant decrease in such level. The decreasecan be, for example, at least 10%, at least 20%, at least 30%, at least40% or more, and is preferably down to a level accepted as within therange of normal for an individual without such disorder.

By “increase” or “raise” in the context of a disease marker or symptomis meant a statistically significant rise in such level. The increasecan be, for example, at least 10%, at least 20%, at least 30%, at least40% or more, and is preferably up to a level accepted as within therange of normal for an individual without such disorder.

As used herein, the phrases “therapeutically effective amount” and“prophylactically effective amount” refer to an amount that provides atherapeutic benefit in the treatment, prevention, or management ofpathological processes or an overt symptom of one or more pathologicalprocesses. The specific amount that is therapeutically effective can bereadily determined by an ordinary medical practitioner, and may varydepending on factors known in the art, such as, for example, the type ofpathological processes, patient history and age, the stage ofpathological processes, and the administration of other agents thatinhibit pathological processes.

As used herein, a “pharmaceutical composition” comprises apharmacologically effective amount of a polypeptide and apharmaceutically acceptable carrier. As used herein, “pharmacologicallyeffective amount,” “therapeutically effective amount” or simply“effective amount” refers to that amount of a polypeptide effective toproduce the intended pharmacological, therapeutic or preventive result.For example, if a given clinical treatment is considered effective whenthere is at least a 10% alteration (increase or decrease) in ameasurable parameter associated with a disease or disorder, atherapeutically effective amount of a drug for the treatment of thatdisease or disorder is the amount necessary to effect at least a 10%alteration in that parameter. For example, a therapeutically effectiveamount of a polypeptide may be one that alters binding of a target toits natural binding partner by at least 10%.

The term “pharmaceutically acceptable carrier” refers to a carrier foradministration of a therapeutic agent. Such carriers include, but arenot limited to, saline, buffered saline, dextrose, water, glycerol,ethanol, and combinations thereof. The term specifically excludes cellculture medium. For drugs administered orally, pharmaceuticallyacceptable carriers include, but are not limited to pharmaceuticallyacceptable excipients such as inert diluents, disintegrating agents,binding agents, lubricating agents, sweetening agents, flavoring agents,coloring agents and preservatives. Suitable inert diluents includesodium and calcium carbonate, sodium and calcium phosphate, and lactose,while corn starch and alginic acid are suitable disintegrating agents.Binding agents may include starch and gelatin, while the lubricatingagent, if present, will generally be magnesium stearate, stearic acid ortalc. If desired, the tablets may be coated with a material such asglyceryl monostearate or glyceryl distearate, to delay absorption in thegastrointestinal tract. Agents included in drug formulations aredescribed further herein below.

Efficacy of treatment or amelioration of disease can be assessed, forexample by measuring disease progression, disease remission, symptomseverity, reduction in pain, quality of life, dose of a medicationrequired to sustain a treatment effect, level of a disease marker or anyother measurable parameter appropriate for a given disease being treatedor targeted for prevention. It is well within the ability of one skilledin the art to monitor efficacy of treatment or prevention by measuringany one of such parameters, or any combination of parameters. Inconnection with the administration of a polypeptide or pharmaceuticalcomposition thereof, “effective against” a disease or disorder indicatesthat administration in a clinically appropriate manner results in abeneficial effect for at least a fraction of patients, such as animprovement of symptoms, a cure, a reduction in disease load, reductionin tumor mass or cell numbers, extension of life, improvement in qualityof life, a reduction in the need for blood transfusions or other effectgenerally recognized as positive by medical doctors familiar withtreating the particular type of disease or disorder.

A treatment or preventive effect is evident when there is astatistically significant improvement in one or more parameters ofdisease status, or by a failure to worsen or to develop symptoms wherethey would otherwise be anticipated. As an example, a favorable changeof at least 10% in a measurable parameter of disease, and preferably atleast 20%, 30%, 40%, 50% or more can be indicative of effectivetreatment. Efficacy for a given polypeptide drug or formulation of thatdrug can also be judged using an experimental animal model for the givendisease as known in the art. When using an experimental animal model,efficacy of treatment is evidenced when a statistically significantmodulation in a marker or symptom is observed.

The polypeptide and an additional therapeutic agent can be administeredin combination in the same composition, e.g., parenterally, or theadditional therapeutic agent can be administered as part of a separatecomposition or by another method described herein.

Inflammatory Indications

In some embodiments, compounds and compositions of the invention may beused to treat subjects with diseases, disorders and/or conditionsrelated to inflammation. Inflammation may be upregulated during theproteolytic cascade of the complement system. Although inflammation mayhave beneficial effects, excess inflammation may lead to a variety ofpathologies (Markiewski et al. 2007. Am J Pathol. 17: 715-27).Accordingly, compounds and compositions of the present invention may beused to reduce or eliminate inflammation associated with complementactivation.

Sterile Inflammation

In some embodiments, compounds and compositions of the present inventionmay be used to treat, prevent or delay development of sterileinflammation. Sterile inflammation is inflammation that occurs inresponse to stimuli other than infection. Sterile inflammation may be acommon response to stress such as genomic stress, hypoxic stress,nutrient stress or endoplasmic reticulum stress caused by a physical,chemical, or metabolic noxious stimuli. Sterile inflammation maycontribute to pathogenesis of many diseases such as, but not limited to,ischemia-induced injuries, rheumatoid arthritis, acute lung injuries,drug-induced liver injuries, inflammatory bowel diseases and/or otherdiseases, disorders or conditions. Mechanism of sterile inflammation andmethods and compositions for treatment, prevention and/or delaying ofsymptoms of sterile inflammation may include any of those taught byRubartelli et al. in Frontiers in Immunology, 2013, 4:398-99, Rock etal. in Annu Rev Immunol. 2010, 28:321-342 or in U.S. Pat. No. 8,101,586,the contents of each of which are herein incorporated by reference intheir entirety.

Systemic Inflammatory Response (SIRS) and Sepsis

In some embodiments, compounds and compositions of the invention may beused to treat and/or prevent systemic inflammatory response syndrome(SIRS). SIRS is inflammation affecting the whole body. Where SIRS iscaused by an infection, it is referred to as sepsis. SIRS may also becaused by non-infectious events such as trauma, injury, burns, ischemia,hemorrhage and/or other conditions. During sepsis and SIRS, complementactivation leads to excessive generation of complement activationproducts which may cause multi organ failure (MOF) in subjects.Compounds and compositions of the invention may be used to controland/or balance complement activation for prevention and treatment ofSIRS, sepsis and/or MOF. The methods of applying complement inhibitorsto treat SIRS and sepsis may include those taught by Rittirsch et al. inClin Dev Immunol, 2012, 962927, in U.S. publication No. US2013/0053302or in U.S. Pat. No. 8,329,169, the contents of each of which are hereinincorporated by reference in their entirety.

Acute Respiratory Distress Syndrome (ARDS)

In some embodiments, compounds and compositions of the invention may beused to treat and/or prevent development of acute respiratory distresssyndrome (ARDS). ARDS is a widespread inflammation of the lungs and maybe caused by trauma, infection (e.g., sepsis), severe pneumonia and/orinhalation of harmful substances. ARDS is typically a severe,life-threatening complication. Studies suggest that neutrophils maycontribute to development of ARDS by affecting the accumulation ofpolymorphonuclear cells in the injured pulmonary alveoli andinterstitial tissue of the lungs. Accordingly, compounds andcompositions of the invention may be administered to reduce and/orprevent tissue factor production in alveolar neutrophils. Compounds andcompositions of the invention may further be used for treatment,prevention and/or delaying of ARDS, in some cases according to any ofthe methods taught in International publication No. WO2009/014633, thecontents of which are herein incorporated by reference in theirentirety.

Periodontitis

In some embodiments, compounds and compositions of the invention may beused to treat or prevent development of periodontitis and/or associatedconditions. Periodontitis is a widespread, chronic inflammation leadingto the destruction of periodontal tissue which is the tissue supportingand surrounding the teeth. The condition also involves alveolar boneloss (bone that holds the teeth). Periodontitis may be caused by a lackof oral hygiene leading to accumulation of bacteria at the gum line,also known as dental plaque. Certain health conditions such as diabetesor malnutrition and/or habits such as smoking may increase the risk ofperiodontitis. Periodontitis may increase the risk of stroke, myocardialinfarction, atherosclerosis, diabetes, osteoporosis, pre-term labor, aswell as other health issues. Studies demonstrate a correlation betweenperiodontitis and local complement activity. Periodontal bacteria mayeither inhibit or activate certain components of the complement cascade.Accordingly, compounds and compositions of the invention may be used toprevent and/or treat periodontitis and associated diseases andconditions. Complement activation inhibitors and treatment methods mayinclude any of those taught by Hajishengallis in Biochem Pharmacol.2010, 15; 80(12): 1 and Lambris or in US publication No. US2013/0344082,the contents of each of which are herein incorporated by reference intheir entirety.

Wounds and Injuries

Compounds and compositions of the invention may be used to treat and/orpromote healing of different types of wounds and/or injuries. As usedherein, the term “injury” typically refers to physical trauma, but mayinclude localized infection or disease processes. Injuries may becharacterized by harm, damage or destruction caused by external eventsaffecting body parts and/or organs. Wounds are associated with cuts,blows, burns and/or other impacts to the skin, leaving the skin brokenor damaged. Wounds and injuries are often acute but if not healedproperly they may lead to chronic complications and/or inflammation.

Wounds and Burn Wounds

In some embodiments, compounds and compositions of the invention may beused to treat and/or to promote healing of wounds. Healthy skin providesa waterproof, protective barrier against pathogens and otherenvironmental effectors. The skin also controls body temperature andfluid evaporation. When skin is wounded these functions are disruptedmaking skin healing challenging. Wounding initiates a set ofphysiological processes related to the immune system that repair andregenerate tissue. Complement activation is one of these processes.Complement activation studies have identified several complementcomponents involved with wound healing as taught by van de Goot et al.in J Burn Care Res 2009, 30:274-280 and Cazander et al. Clin DevImmunol, 2012, 2012:534291, the contents of each of which are hereinincorporated by reference in their entirety. In some cases, complementactivation may be excessive, causing cell death and enhancedinflammation (leading to impaired wound healing and chronic wounds). Insome cases, compounds and compositions of the present invention may beused to reduce or eliminate such complement activation to promote woundhealing. Treatment with compounds and compositions of the invention maybe carried out according to any of the methods for treating woundsdisclosed in International publication number WO2012/174055, thecontents of which are herein incorporated by reference in theirentirety.

Head Trauma

In some embodiments, compounds and compositions of the invention may beused to treat and/or promote healing of head trauma. Head traumasinclude injuries to the scalp, the skull or the brain. Examples of headtrauma include, but are not limited to concussions, contusions, skullfracture, traumatic brain injuries and/or other injuries. Head traumasmay be minor or severe. In some cases, head trauma may lead to long termphysical and/or mental complications or death. Studies indicate thathead traumas may induce improper intracranial complement cascadeactivation, which may lead to local inflammatory responses contributingto secondary brain damage by development of brain edema and/or neuronaldeath (Stahel et al. in Brain Research Reviews, 1998, 27: 243-56, thecontents of which are herein incorporated by reference in theirentirety). Compounds and compositions of the invention may be used totreat head trauma and/or to reduce or prevent related secondarycomplications. Methods of using compounds and compositions of theinvention to control complement cascade activation in head trauma mayinclude any of those taught by Holers et al. in U.S. Pat. No. 8,911,733,the contents of which are herein incorporated by reference in theirentirety.

Crush Injury

In some embodiments, compounds and compositions of the invention may beused to treat and/or promote healing of crush injuries. Crush injuriesare injuries caused by a force or a pressure put on the body causingbleeding, bruising, fractures, nerve injuries, wounds and/or otherdamages to the body. Compounds and compositions of the invention may beused to reduce complement activation following crush injuries, therebypromoting healing after crush injuries (e.g. by promoting nerveregeneration, promoting fracture healing, preventing or treatinginflammation, and/or other related complications). Compounds andcompositions of the invention may be used to promote healing accordingto any of the methods taught in U.S. Pat. No. 8,703,136; InternationalPublication Nos. WO2012/162215; WO2012/174055; or US publication No.US2006/0270590, the contents of each of which are herein incorporated byreference in their entirety.

Autoimmune Disease

The compounds and compositions of the invention may be used to treatsubjects with autoimmune diseases and/or disorders. The immune systemmay be divided into innate and adaptive systems, referring tononspecific immediate defense mechanisms and more complexantigen-specific systems, respectively. The complement system is part ofthe innate immune system, recognizing and eliminating pathogens.Additionally, complement proteins may modulate adaptive immunity,connecting innate and adaptive responses. Autoimmune diseases anddisorders are immune abnormalities causing the system to target selftissues and substances. Autoimmune disease may involve certain tissuesor organs of the body. Compounds and compositions of the invention maybe used to modulate complement in the treatment and/or prevention ofautoimmune diseases. In some cases, such compounds and compositions maybe used according to the methods presented in Ballanti et al. ImmunolRes (2013) 56:477-491, the contents of which are herein incorporated byreference in their entirety.

Anti-Phospholipid Syndrome (APS) and Catastrophic Anti-PhospholipidSyndrome (CAPS)

In some embodiments, compounds and compositions of the invention may beused to prevent and/or treat anti-phospholipid syndrome (APS) bycomplement activation control. APS is an autoimmune condition caused byanti-phospholipid antibodies that cause the blood to clot. APS may leadto recurrent venous or arterial thrombosis in organs, and complicationsin placental circulations causing pregnancy-related complications suchas miscarriage, still birth, preeclampsia, premature birth and/or othercomplications. Catastrophic anti-phospholipid syndrome (CAPS) is anextreme and acute version of a similar condition leading to occlusion ofveins in several organs simultaneously. Studies suggest that complementactivation may contribute to APS-related complications includingpregnancy-related complications, thrombotic (clotting) complications,and vascular complications. Compound and compositions of the inventionmay be used to treat APS-related conditions by reducing or eliminatingcomplement activation. In some cases, compounds and compositions of theinvention may be used to treat APS and/or APS-related complicationsaccording to the methods taught by Salmon et al. Ann Rheum Dis 2002;61(Suppl II):ii46-ii50 and Mackworth-Young in Clin Exp Immunol 2004,136:393-401, the contents of which are herein incorporated by referencein their entirety.

Cold Agglutinin Disease

In some embodiments, compounds and compositions of the invention may beused to treat cold agglutinin disease (CAD), also referred to as coldagglutinin-mediated hemolysis. CAD is an autoimmune disease resultingfrom a high concentration of IgM antibodies interacting with red bloodcells at low range body temperatures [Engelhardt et al. Blood, 2002,100(5): 1922-23]. CAD may lead to conditions such as anemia, fatigue,dyspnea, hemoglobinuria and/or acrocyanosis. CAD is related to robustcomplement activation and studies have shown that CAD may be treatedwith complement inhibitor therapies. Accordingly, the present inventionprovides methods of treating CAD using compounds and compositions of theinvention. In some cases, compounds and compositions of the inventionmay be used to treat CAD according to the methods taught by Roth et alin Blood, 2009, 113:3885-86 or in International publication No.WO2012/139081, the contents of each of which are herein incorporated byreference in their entirety.

Vascular Indications

In some embodiments, compounds and compositions of the invention may beused to treat vascular indications affecting blood vessels (e.g.,arteries, veins, and capillaries). Such indications may affect bloodcirculation, blood pressure, blood flow, organ function and/or otherbodily functions.

Thrombotic Microangiopathy (TMA)

In some embodiments, compounds and compositions of the invention may beused to treat and/or prevent thrombotic microangiopathy (TMA) andassociated diseases. Microangiopathies affect small blood vessels(capillaries) of the body causing capillary walls to become thick, weak,and prone to bleeding and slow blood circulation. TMAs tend to lead tothe development of vascular thrombi, endothelial cell damage,thrombocytopenia, and hemolysis. Organs such as the brain, kidney,muscles, gastrointestinal system, skin, and lungs may be affected. TMAsmay arise from medical operations and/or conditions that include, butare not limited to, hematopoietic stem cell transplantation (HSCT),renal disorders, diabetes and/or other conditions. TMAs may be caused byunderlying complement system dysfunction, as described by Meri et al. inEuropean Journal of Internal Medicine, 2013, 24: 496-502, the contentsof which are herein incorporated by reference in their entirety.Generally, TMAs may result from increased levels of certain complementcomponents leading to thrombosis. In some cases, this may be caused bymutations in complement proteins or related enzymes. Resultingcomplement dysfunction may lead to complement targeting of endothelialcells and platelets leading to increased thrombosis. In someembodiments, TMAs may be prevented and/or treated with compounds andcompositions of the invention. In some cases, methods of treating TMAswith compounds and compositions of the invention may be carried outaccording to those described in US publication Nos. US2012/0225056 orUS2013/0246083, the contents of each of which are herein incorporated byreference in their entirety.

Disseminated Intravascular Coagulation (DIC)

In some embodiments, compounds and compositions of the invention may beused to prevent and/or treat disseminated intravascular coagulation(DIC) by controlling complement activation. DIC is a pathologicalcondition where the clotting cascade in blood is widely activated andresults in formation of blood clots especially in the capillaries. DICmay lead to an obstructed blood flow of tissues and may eventuallydamage organs. Additionally, DIC affects the normal process of bloodclotting that may lead to severe bleeding. Compounds and compositions ofthe invention may be used to treat, prevent or reduce the severity ofDIC by modulating complement activity. In some cases compounds andcompositions of the invention may be used according to any of themethods of DIC treatment taught in U.S. Pat. No. 8,652,477, the contentsof which are herein incorporated by reference in their entirety.

Vasculitis

In some embodiments, compounds and compositions of the invention may beused to prevent and/or treat vasculitis. Generally, vasculitis is adisorder related to inflammation of blood vessels, including veins andarteries, characterized by white blood cells attacking tissues andcausing swelling of the blood vessels. Vasculitis may be associated withan infection, such as in Rocky Mountain spotted fever, or autoimmunity.An example of autoimmunity associated vasculitis is Anti-NeutrophilCytoplasmic Autoantibody (ANCA) vasculitis. ANCA vasculitis is caused byabnormal antibodies attacking the body's own cells and tissues. ANCAsattack the cytoplasm of certain white blood cells and neutrophils,causing them to attack the walls of the vessels in certain organs andtissues of the body. ANCA vasculitis may affect skin, lungs, eyes and/orkidney. Studies suggest that ANCA disease activates an alternativecomplement pathway and generates certain complement components thatcreate an inflammation amplification loop resulting in a vascular injury(Jennette et al. 2013, Semin Nephrol. 33(6): 557-64, the contents ofwhich are herein incorporated by reference in their entirety). In somecases, compounds and compositions of the invention may be used toprevent and/or treat ANCA vasculitis by inhibiting complementactivation.

Neurological Indications

The compounds and compositions of the invention may be used to prevent,treat and/or ease the symptoms of neurological indications, including,but not limited to neurodegenerative diseases and related disorders.Neurodegeneration generally relates to a loss of structure or functionof neurons, including death of neurons. These disorders may be treatedby inhibiting the effect of complement on neuronal cells using compoundsand compositions of the invention. Neurodegenerative related disordersinclude, but are not limited to, Amyelotrophic Lateral Sclerosis (ALS),Multiple Sclerosis (MS), Parkinson's disease and Alzheimer's disease.

Amyotrophic Lateral Sclerosis (ALS)

In some embodiments, compounds and compositions of the invention may beused to prevent, treat and/or ease the symptoms of ALS. ALS is a fatalmotor neuron disease characterized by the degeneration of spinal cordneurons, brainstems and motor cortex. ALS causes loss of muscle strengthleading eventually to a respiratory failure. Complement dysfunction maycontribute to ALS, and therefore ALS may be prevented, treated and/orthe symptoms may be reduced by therapy with compounds and compositionsof the invention targeting complement activity. In some cases, compoundsand compositions of the invention may be used to promote nerveregeneration. In some cases, compounds and compositions of the inventionmay be used as complement inhibitors according to any of the methodstaught in US publication No. US2014/0234275 or US2010/0143344, thecontents of each of which are herein incorporated by reference in theirentirety.

Alzheimer's Disease

In some embodiments, compounds and compositions of the invention may beused to prevent and/or treat Alzheimer's disease by controllingcomplement activity. Alzheimer's disease is a chronic neurodegenerativedisease with symptoms that may include disorientation, memory loss, moodswings, behavioral problems and eventually loss of bodily functions.Alzheimer's disease is thought to be caused by extracellular braindeposits of amyloid that are associated with inflammation-relatedproteins such as complement proteins (Sjoberg et al. 2009. Trends inImmunology. 30(2): 83-90, the contents of which are herein incorporatedby reference in their entirety). In some cases, compounds andcompositions of the invention may be used as complement inhibitorsaccording to any of the Alzheimer's treatment methods taught in USpublication No. US2014/0234275, the contents of which are hereinincorporated by reference in their entirety.

Kidney-Related Indications

The compounds and compositions of the invention may be used to treatcertain diseases, disorders and/or conditions related to kidneys, insome cases by inhibiting complement activity. Kidneys are organsresponsible for removing metabolic waste products from the blood stream.Kidneys regulate blood pressure, the urinary system, and homeostaticfunctions and are therefore essential for a variety of bodily functions.Kidneys may be more seriously affected by inflammation (as compared toother organs) due to unique structural features and exposure to blood.Kidneys also produce their own complement proteins which may beactivated upon infection, kidney disease, and renal transplantations. Insome cases, compounds and compositions of the invention may be used ascomplement inhibitors in the treatment of certain diseases, conditions,and/or disorders of the kidney according to the methods taught by Quigg,J Immunol 2003; 171:3319-24, the contents of which are hereinincorporated by reference in their entirety.

Lupus Nephritis

In some embodiments, compounds and compositions of the invention may beused to prevent and/or treat lupus nephritis by inhibiting complementactivity. Lupus nephritis is a kidney inflammation caused by anautoimmune disease called systemic lupus erythematosus (SLE). Symptomsof lupus nephritis include high blood pressure; foamy urine; swelling ofthe legs, the feet, the hands, or the face; joint pain; muscle pain;fever; and rash. Lupus nephritis may be treated by inhibitors thatcontrol complement activity, including compounds and compositions of thepresent invention. Methods and compositions for preventing and/ortreating Lupus nephritis by complement inhibition may include any ofthose taught in US publication No. US2013/0345257 or U.S. Pat. No.8,377,437, the contents of each of which are herein incorporated byreference in their entirety.

Membranous Glomerulonephritis (MGN)

In some embodiments, compounds and compositions of the invention may beused to prevent and/or treat membranous glomerulonephritis (MGN)disorder by inhibiting the activation of certain complement components.MGN is a disorder of the kidney that may lead to inflammation andstructural changes. MGN is caused by antibodies binding to a solubleantigen in kidney capillaries (glomerulus). MGN may affect kidneyfunctions, such as filtering fluids and may lead to kidney failure.Compounds and compositions of the invention may be used according tomethods of preventing and/or treating MGN by complement inhibitiontaught in U.S. publication No. US2010/0015139 or in Internationalpublication No. WO2000/021559, the contents of each of which are hereinincorporated by reference in their entirety.

Hemodialysis Complications

In some embodiments, compounds and compositions of the invention may beused to prevent and/or treat complications associated with hemodialysisby inhibiting complement activation. Hemodialysis is a medical procedureused to maintain kidney function in subjects with kidney failure. Inhemodialysis, the removal of waste products such as creatinine, urea,and free water from blood is performed externally. A common complicationof hemodialysis treatment is chronic inflammation caused by contactbetween blood and the dialysis membrane. Another common complication isthrombosis referring to a formation of blood clots that obstructs theblood circulation. Studies have suggested that these complications arerelated to complement activation. Hemodialysis may be combined withcomplement inhibitor therapy to provide means of controllinginflammatory responses and pathologies and/or preventing or treatingthrombosis in subjects going through hemodialysis due to kidney failure.Methods of using compounds and compositions of the invention fortreatment of hemodialysis complications may be carried out according toany of the methods taught by DeAngelis et al in Immunobiology, 2012,217(11): 1097-1105 or by Kourtzelis et al. Blood, 2010, 116(4):631-639,the contents of each of which are herein incorporated by reference intheir entirety.

Ocular Diseases

In some embodiments, compounds and compositions of the invention may beused to prevent and/or treat certain ocular related diseases, disordersand/or conditions. In a healthy eye the complement system is activatedat a low level and is continuously regulated by membrane-bound andsoluble intraocular proteins that protect against pathogens. Thereforethe activation of complement plays an important role in severalcomplications related to the eye and controlling complement activationmay be used to treat such diseases. Compounds and compositions of theinvention may be used as complement inhibitors in the treatment ofocular disease according to any of the methods taught by Jha et al. inMol Immunol. 2007; 44(16): 3901-3908 or in U.S. Pat. No. 8,753,625, thecontents of each of which are herein incorporated by reference in theirentirety.

Age-Related Macular Degeneration (AMD)

In some embodiments, compounds and compositions of the invention may beused to prevent and/or treat age-related macular degeneration (AMD) byinhibiting ocular complement activation. AMD is a chronic ocular diseasecausing blurred central vision, blind spots in central vision, and/oreventual loss of central vision. Central vision affects ability to read,drive a vehicle and/or recognize faces. AMD is generally divided intotwo types, non-exudative (dry) and exudative (wet). Dry AMD refers tothe deterioration of the macula which is the tissue in the center of theretina. Wet AMD refers to the failure of blood vessels under the retinaleading to leaking of blood and fluid. Several human and animal studieshave identified complement proteins that are related to AMD and noveltherapeutic strategies included controlling complement activationpathways, as discussed by Jha et al. in Mol Immunol. 2007; 44(16):3901-8. Methods of the invention involving the use of compounds andcompositions of the invention for prevention and/or treatment of AMD mayinclude any of those taught in US publication Nos. US2011/0269807 orUS2008/0269318, the contents of each of which are herein incorporated byreference in their entirety.

Corneal Disease

In some embodiments, compounds and compositions of the invention may beused to prevent and/or treat corneal diseases by inhibiting ocularcomplement activation. The complement system plays an important role inprotection of the cornea from pathogenic particles and/or inflammatoryantigens. The cornea is the outermost front part of the eye covering andprotecting the iris, pupil and anterior chamber and is therefore exposedto external factors. Corneal diseases include, but are not limited to,keratoconus, keratitis, ocular herpes and/or other diseases. Cornealcomplications may cause pain, blurred vision, tearing, redness, lightsensitivity and/or corneal scarring. The complement system is criticalfor corneal protection, but complement activation may cause damage tothe corneal tissue after an infection is cleared as certain complementcompounds are heavily expressed. Methods of the present invention formodulating complement activity in the treatment of corneal disease mayinclude any of those taught by Jha et al. in Mol Immunol. 2007; 44(16):3901-8, the contents of which are herein incorporated by reference intheir entirety.

Autoimmune Uveitis

In some embodiments, compounds and compositions of the invention may beused to prevent and/or treat uveitis, which is an inflammation of theuveal layer of the eye. Uvea is the pigmented area of the eye comprisingthe choroids, iris and ciliary body of the eye. Uveitis causes redness,blurred vision, pain, synechia and may eventually cause blindness.Studies have indicated that complement activation products are presentin the eyes of patients with autoimmune uveitis and complement plays animportant role in disease development. In some cases, compounds andcompositions of the invention may be used to treat and/or preventuveitis according to any of the methods identified in Jha et al. in MolImmunol. 2007. 44(16): 3901-8, the contents of which are hereinincorporated by reference in their entirety.

Diabetic Retinopathy

In some embodiments, compounds and compositions of the invention may beused to prevent and/or treat diabetic retinopathy which is a diseasecaused by changes in retinal blood vessels in diabetic patients.Retinopathy may cause blood vessel swelling and fluid leaking and/orgrowth of abnormal blood vessels. Diabetic retinopathy affects visionand may eventually lead to blindness. Studies have suggested thatactivation of complement has an important role in the development ofdiabetic retinopathy. In some cases, compounds and compositions of theinvention may be used according to methods of diabetic retinopathytreatment described in Jha et al. Mol Immunol. 2007; 44(16): 3901-8, thecontents of which are herein incorporated by reference in theirentirety.

Pre-Eclampsia and HELLP-Syndrome

In some embodiments, compounds and compositions of the invention may beused to prevent and/or treat pre-eclampsia and/or HELLP (abbreviationstanding for syndrome features of 1) hemolysis, 2) elevated liverenzymes and 3) low platelet count) syndrome by complement inhibitortherapy. Pre-eclampsia is a disorder of pregnancy with symptomsincluding elevated blood pressure, swelling, shortness of breath, kidneydysfunction, impaired liver function and/or low blood platelet count.Pre-eclampsia is typically diagnosed by a high urine protein level andhigh blood pressure. HELLP syndrome is a combination of hemolysis,elevated liver enzymes and low platelet conditions. Hemolysis is adisease involving rupturing of red blood cells leading to the release ofhemoglobin from red blood cells. Elevated liver enzymes may indicate apregnancy-induced liver condition. Low platelet levels lead to reducedclotting capability, causing danger of excessive bleeding. HELLP isassociated with a pre-eclampsia and liver disorder. HELLP syndrometypically occurs during the later stages of pregnancy or afterchildbirth. It is typically diagnosed by blood tests indicating thepresence of the three conditions it involves. Typically HELLP is treatedby inducing delivery.

Studies suggest that complement activation occurs during HELLP syndromeand pre-eclampsia and that certain complement components are present atincreased levels during HELLP and pre-eclampsia. Complement inhibitorsmay be used as therapeutic agents to prevent and/or treat theseconditions. Compounds and compositions of the invention may be usedaccording to methods of preventing and/or treating HELLP andpre-eclampsia taught by Heager et al. in Obstetrics & Gynecology, 1992,79(1):19-26 or in International publication No. WO201/078622, thecontents of each of which are herein incorporated by reference in theirentirety.

Dosage and Administration

For use as treatment of human subjects, polypeptides can be formulatedas pharmaceutical compositions. Depending on the subject to be treated,the mode of administration, and the type of treatment desired (e.g.,prevention, prophylaxis, or therapy) the polypeptides are formulated inways consonant with these parameters. A summary of such techniques isfound in Remington: The Science and Practice of Pharmacy, 21st Edition,Lippincott Williams & Wilkins, (2005); and Encyclopedia ofPharmaceutical Technology, eds. J. Swarbrick and J. C. Boylan,1988-1999, Marcel Dekker, New York, each of which is incorporated hereinby reference.

Compositions of the present invention are preferably provided in atherapeutically effective amount, which may be, for example, a dailyamount of from about 0.1 mg to about 100 mg, from about 0.5 mg to about200 mg, from about 1 mg to about 300 mg, from about 5 mg to about 500mg, from about 10 mg to about 750 mg, from about 50 mg to about 1000 mgor at least 1000 mg. In one embodiment, a pharmaceutical compositioncomprises a capsule, for example in unit dosage form.

According to some methods of the invention, compounds and compositionsof the invention are provided at concentrations needed to achieve adesired effect. In some cases, compounds and compositions of theinvention are provided at an amount necessary to reduce a given reactionor process by half. The concentration needed to achieve such a reductionis referred to herein as the half maximal inhibitory concentration, or“IC₅₀.” Alternatively, compounds and compositions of the invention maybe provided at an amount necessary to increase a given reaction,activity or process by half. The concentration needed for such anincrease is referred to herein as the half maximal effectiveconcentration of “EC₅₀.”

Unit Dosage Forms

The polypeptides of the invention may be present in amounts totaling0.1-95% by weight of the total weight of the composition. Thecomposition may be provided in a dosage form that is suitable for oraladministration. Thus, the pharmaceutical composition may be in the formof, e.g., hard capsules (e.g., hard gelatin capsules or hardhydroxypropyl methylcellulose capsules), soft gelatin capsules, tablets,caplets, enteric coated tablets, chewable tablets, enteric coated hardgelatin capsules, enteric coated soft gelatin capsules, minicapsules,lozenges, films, strips, gelcaps, dragees, solutions, emulsions,suspensions, syrups, or sprays.

Subjects may be administered a therapeutic amount of a polypeptide,including, but not limited to doses of 0.01 mg/kg, 1.0 mg/kg, or 15mg/kg. In some cases, polypeptides of the invention are administered atfrom about 0.001 mg/kg to about 1.0 mg/kg, from about 0.01 mg/kg toabout 2.0 mg/kg, from about 0.05 mg/kg to about 5.0 mg/kg, from about0.03 mg/kg to about 3.0 mg/kg, from about 0.01 mg/kg to about 10 mg/kg,from about 1.0 mg/kg to about 5.0 mg/kg, from about 2.0 mg/kg to about4.0 mg/kg, from about 1.5 mg/kg to about 7.5 mg/kg, from about 5.0 mg/kgto about 15 mg/kg, from about 7.5 mg/kg to about 12.5 mg/kg, or fromabout 10 mg/kg to about 20 mg/kg. Such ranges include ranges suitablefor administration to human subjects. In some cases, human subjects maybe administered from about 0.01 mg/kg to about 10 mg/kg, from about 0.01mg/kg to about 15 mg/kg, or from about 3 mg/kg to about 5 mg/kg. Dosagelevels may be highly dependent on the nature of the condition; drugefficacy; the condition of the patient; the judgment of thepractitioner; and the frequency and mode of administration.

In some cases, polypeptides of the invention are provided atconcentrations adjusted to achieve a desired level of such polypeptidesin a sample, biological system, or subject (e.g., plasma level in asubject). In some cases, desired concentrations of polypeptides in asample, biological system or subject may include concentrations of fromabout 0.001 nM to about 0.01 nM, from about 0.005 nM to about 0.05 nM,from about 0.02 nM to about 0.2 nM, from about 0.03 nM to about 0.3 nM,from about 0.05 nM to about 0.5 nM, from about 0.01 nM to about 2.0 nM,from about 0.1 nM to about 50 nM, from about 0.1 nM to about 10 nM, fromabout 0.1 nM to about 5 nM, or from about 0.2 nM to about 20 nM. In somecases, desired concentrations of polypeptides in subject plasma may befrom about 0.01 mg/L to about 2 mg/L, from about 0.02 mg/L to about 4mg/L, from about 0.05 mg/L to about 5 mg/L, from about 0.1 mg/L to about1.0 mg/L, from about 0.2 mg/L to about 2.0 mg/L, from about 0.5 mg/L toabout 5 mg/L, from about 1 mg/L to about 5 mg/L, from about 2 mg/L toabout 10 mg/L, from about 3 mg/L to about 9 mg/L, or from about 5 mg/Lto about 20 mg/L.

In other embodiments, the polypeptides are administered at a frequencyof e.g., every 4 hr, every 6 hr, every 12 hr, every 18 hr, every 24 hr,every 36 hr, every 72 hr, every 84 hr, every 96 hr, every 5 days, every7 days, every 10 days, every 14 days, every 3 weeks, or more. Thecompositions can be administered once daily or the polypeptide can beadministered as two, three, or more sub-doses at appropriate intervalsthroughout the day or delivery through a controlled release formulation.In that case, the polypeptide contained in each sub-dose must becorrespondingly smaller in order to achieve the total daily dosage. Thedosage unit can also be compounded for delivery over several days, e.g.,using a conventional sustained release formulation, which providessustained release of the polypeptide over a several-day-period.

Sustained release formulations are well known in the art and areparticularly useful for delivery of agents to a particular site, such ascould be used with the polypeptide compositions of the presentinvention. The effect of a single dose can be long-lasting, such thatsubsequent doses are administered at not more than 3-, 4-, or 5-dayintervals, or at not more than 1, 2-, 3-, or 4-week intervals.

The polypeptide can be administered by intravenous infusion over aperiod of time, such as over a 5 minute, 10 minute, 15 minute, 20minute, or 25 minute period. The administration may be repeated, forexample, on a regular basis, such as biweekly (i.e., every two weeks)for one month, two months, three months, four months or longer. After aninitial treatment regimen, the treatments can be administered on a lessfrequent basis. For example, after administration biweekly for threemonths, administration can be repeated once per month, for six months ora year or longer. Administration of the polypeptide or composition canreduce, lower, increase or alter binding or any physiologicallydeleterious process, e.g., in a cell, tissue, blood, urine or othercompartment of a patient by at least 10%, at least 15%, at least 20%, atleast 25%, at least 30%, at least 40%, at least 50%, at least 60%, atleast 70%, at least 80% or at least 90% or more.

Before administration of a full dose of the polypeptide and/orpolypeptide composition, patients can be administered a smaller dose,such as 5% of a full dose, and monitored for adverse effects, such as anallergic reaction or infusion reaction, or for elevated lipid levels orblood pressure. In another example, the patient can be monitored forunwanted immunostimulatory effects, such as increased cytokine (e.g.,TNF-alpha, Il-1, Il-6, or Il-10) levels.

Genetic predisposition plays a role in the development of some diseasesor disorders. Therefore, a patient in need of a polypeptide and/orpolypeptide composition may be identified by taking a family history,or, for example, screening for one or more genetic markers or variants.A healthcare provider, such as a doctor, nurse, or family member, cantake a family history before prescribing or administering a therapeuticcomposition of the present invention.

III. Kits

Any of the compositions described herein may be comprised in a kit. In anon-limiting example, polypeptides may be included in a kit for treatinga disease. The kit may include a vial of sterile, dry polypeptidepowder, sterile solution for dissolving the dried powder, and a syringefor infusion set for administering the polypeptide.

When polypeptides are provided as a dried powder it is contemplated thatbetween 10 micrograms and 1000 milligrams of polypeptide, or at least orat most those amounts are provided in kits of the invention

The container means will generally include at least one vial, test tube,flask, bottle, syringe and/or other container means, into which thepolypeptide formulations are placed, preferably, suitably allocated. Thekits may also comprise a second container means for containing asterile, pharmaceutically acceptable buffer and/or other diluent.

A kit can include instructions for employing the kit components as wellthe use of any other reagent not included in the kit. Instructions mayinclude variations that can be implemented.

While various embodiments of the invention have been particularly shownand described, it will be understood by those skilled in the art thatvarious changes in form and details may be made therein withoutdeparting from the spirit and scope of the invention as defined by theappended claims.

EQUIVALENTS AND SCOPE

Those skilled in the art will recognize, or be able to ascertain usingno more than routine experimentation, many equivalents to the specificembodiments in accordance with the invention described herein. The scopeof the present invention is not intended to be limited to the aboveDescription, but rather is as set forth in the appended claims.

In the claims, articles such as “a,” “an,” and “the” may mean one ormore than one unless indicated to the contrary or otherwise evident fromthe context. Claims or descriptions that include “or” between one ormore members of a group are considered satisfied if one, more than one,or all of the group members are present in, employed in, or otherwiserelevant to a given product or process unless indicated to the contraryor otherwise evident from the context. The invention includesembodiments in which exactly one member of the group is present in,employed in, or otherwise relevant to a given product or process. Theinvention includes embodiments in which more than one, or all of thegroup members are present in, employed in, or otherwise relevant to agiven product or process.

It is also noted that the term “comprising” is intended to be open andpermits but does not require the inclusion of additional elements orsteps. When the term “comprising” is used herein, the terms “consistingof” and “or including” are thus also encompassed and disclosed.

Where ranges are given, endpoints are included. Furthermore, it is to beunderstood that unless otherwise indicated or otherwise evident from thecontext and understanding of one of ordinary skill in the art, valuesthat are expressed as ranges can assume any specific value or subrangewithin the stated ranges in different embodiments of the invention, tothe tenth of the unit of the lower limit of the range, unless thecontext clearly dictates otherwise.

In addition, it is to be understood that any particular embodiment ofthe present invention that falls within the prior art may be explicitlyexcluded from any one or more of the claims. Since such embodiments aredeemed to be known to one of ordinary skill in the art, they may beexcluded even if the exclusion is not set forth explicitly herein. Anyparticular embodiment of the compositions of the invention (e.g., anynucleic acid or protein encoded thereby; any method of production; anymethod of use; etc.) can be excluded from any one or more claims, forany reason, whether or not related to the existence of prior art.

All cited sources, for example, references, publications, databases,database entries, and art cited herein, are incorporated into thisapplication by reference, even if not expressly stated in the citation.In case of conflicting statements of a cited source and the instantapplication, the statement in the instant application shall control.

Section and table headings are not intended to be limiting.

EXAMPLES Example 1. Preparation of Biotinylated C5

An effective final molar ratio of 1:4 C5 to biotin was used for largescale biotinylation. A 10 mM solution of EZ-Link Sulfo-NHS-LC Biotin(Thermo Scientific, Billerica, Mass.) was prepared as per themanufacturer's instructions. To 1 mg of 1 mg/ml C5 (Complement Tech,Tyler Tex.), 2.1 μl of the 10 mM biotin solution was added and incubatedon ice for 2 hours. The reaction was quenched for 30 minutes at 4° C.after the addition of 100 μl of 1 M Tris HCl pH 7.5. The reaction wasdialyzed overnight against cold PBST (phosphate buffered saline(PBS)+0.1% Tween 80). The biotinylated-C5 was aliquoted and stored at−80° C. Biotinylated C5 was characterized by SDS-PAGE under reducing andnon-reducing conditions and characterized for activity by a red bloodcell hemolysis assay. Biotinylated C5 was also checked for recovery onstreptavidin beads (Invitrogen, Grand Island, N.Y.). Capture wasperformed using conditions recommended by the manufacturer. To capture 4μg of biotinylated-C5 from a 100 nM solution, 40 μl of bead slurry wasused and incubated at 4° C. for 1 hour. The concentration of thecaptured biotinylated C5 was calculated by running a known amount of C5on a NuPage 4-12% Bis-Tris gel (Invitrogen, Grand Island, N.Y.).

Example 2. Human Hemolysis Assay for QC of Biotinylated C5

A hemolysis assay was performed with C5-depleted sera and biotinylatedC5 and non-biotinylated C5 to compare the lysis activities of C5 beforeand after biotinylation. Antibody-sensitized sheep erythrocytes(Complement Technology, Tyler Tex.) in solution at 5×10 cell/ml werecentrifuged at 2,090×gravity for 3 minutes and resuspended in GVB++buffer (Complement Technology, Tyler Tex.). C5-depleted human sera(Complement Technology, Tyler Tex.) was rapidly thawed at 37° C. andplaced on ice until diluted in GVB++. Non-biotinylated C5 protein(Complement Technology, Tyler Tex.) and biotinylated C5 protein(in-house biotinylation) was rapidly thawed at 37° C. and placed on awet ice slurry until diluted in GVB++. 100 μl of cells (at a finalconcentration of 2.5×10⁷ cells/ml) was combined with C5-depleted humansera and 50 μl biotinylated C5 or non-biotinylated C5 (with finalconcentrations of either 10 μg/ml, 3 μg/ml or 1 μg/ml) in a tissueculture-treated clear 96-well microtitre plate (USA Scientific, Ocala,Fla.). The plate was incubated for 1 hour at 37° C. After incubation,plates were then centrifuged at 2,090×gravity for 2 minutes beforetransferring 100 μl of supernatant to a new microtitre plate. Absorbancewas read at 412 nm and percent lysis activity of non-biotinylated C5 andbiotinylated C5 was compared.

Example 3. Selection of Polypeptides Binding C5

C5 inhibitors were identified through several rounds of mRNA display andselection. mRNA display was performed generally as described (Roberts,R. W., and Szostak, J. W. (1997). Proc. Natl. Acad. Sci. USA 94,12297-12302; WO2009067191; herein incorporated by reference in itsentirety) with modifications as described herein. RNA pools, weregenerated by in vitro transcription from DNA synthesized with fixedN-terminal methionine and cysteine codons, followed by three positionsof a sixteen codon phosphoramidite mixture, followed by eight positionsof a second codon mixture also containing the cysteine codon. Theresulting mRNA library has a fixed initiating methionine followed by acysteine residue, followed by three positions lacking cysteine, followedby eight positions in which cysteine occurs with a frequency of 12.5%.To conduct the selection, the first round of enrichment comprised afirst step in which RNA pools containing a 3′ terminal UV cross-linkedoligonucleotide containing puromycin were translated in vitro with thepurified translation components listed in Table 2. Translation wascarried out under two separate conditions to generate two uniquelibraries based on amino acid variation. The first condition utilizedonly the 20 natural amino acids while the second condition utilizednatural amino acids (0.1 mM of histidine, threonine, proline, lysine,asparagine, tyrosine, glutamic acid and cysteine), unnatural amino acids(2 mM tertbutyl-glycine (Tbg), 0.8 mM 7-azatryptophan (abbreviated by“azaTrp” in this example) and 1 mM norvaline (Nvl), azaleucine andphenyl-glycine (Phg)) and N-methyl amino acids (450 μM mix ofN-methylated serine [(N-Me)S], alanine [(N-Me)A], glycine [(N-Me)G] and4-fluoro-N-methylphenylalanine [(N-Me-4-F)Phe]). ³⁵S-labeled cysteineresidues were included in both conditions to enable monitoring ofpolypeptide enrichment per round.

TABLE 2 In vitro translation components Component Conc. Creatinephosphate 20 mM MeTHF, pH 7.6 15 μg/ml HEPES-KOH, pH 7.6 51 mM KCl 101mM Spermidine 2 mM DTT 1 mM Creatine kinase 4 mM Myokinase 3 mMNucleotide diphosphate kinase 1 mM Pyrophosphate 1 mM ATP + GTP′ 2 mMeach EF-Tu 50 μM Ribosomes 1 μM MTF 0.56 μM 1F1 0.96 μM IF2 0.40 μM IF30.44 μM EF-G 0.64 μM EF-Ts 1.58 μM RF1 0.24 μM RF3 0.17 μM RRF 0.46 μMMg 17.46 mM

The tRNAs were enzymatically charged with their respective amino acidsusing tRNA synthetases. The four N-methyl tRNAs were pre-charged,whereas all other tRNAs were enzymatically charged during the in vitrotranslation reaction. The tRNA synthetases were added on a volume basisirrespective of their concentrations. 0.1 μl of each tRNA synthetase(except for methionine tRNA synthetase, which was added at 0.4 μl per 25μl translation reaction) was added for in-situ charging duringtranslation for a 25 μl translation reaction. Cross-linked mRNA wasadded at a final concentration of 0.75 μM. The translation reaction waskept at 37° C. for 1 hour. After translation, the fusion of thetranslated polypeptides to their respective mRNAs was carried out byadding high salt to the translation mix and incubating at 37° C. for 1.5hours. A library for the selection of natural polypeptides was preparedfrom eight individual libraries with a fixed cysteine codon in positions5-11. The random positions in these libraries, with all 20 amino acidspossible, were made combinatorially with repeating codon units of NNS (Nis A,G,C, or T; S is G or C) (Devlin, J. J., et. al., (1990). Science249, 404-406.) The translation of these libraries into naturalpolypeptides was done using a rabbit reticulocyte in vitro translationkit rather than the reconstituted system described above.

Recovery of the mRNA-displayed polypeptides was done using both Oligo dTand Ni-NTA affinity, to isolate fusion molecules containing both polyAmRNA and His tagged polypeptides. Oligo dT bead-bound polypeptides werethen cyclized with dibromoxylene as described by others (J. Am. Chem.Soc. 127:1 1727 (2005)).

Direct selection of the polypeptides by target affinity was thenperformed. mRNA-displayed polypeptides were allowed to bind for 1 hourat 4° C. to biotinylated C5 in a 100 nM solution of biotinylated C5 inPBST. The RNA corresponding to the affinity selected polypeptides wasreverse transcribed and PCR amplified to create a double-stranded DNApool. The DNA pool was in vitro transcribed to generate mRNA, and themRNA produced was cross-linked as before at its 3′ terminus with apuromycin-containing oligonucleotide. The mRNA-puromycin fusions weresubjected to in vitro translation to generate the second round of thelibrary, which is now enriched in polypeptides that bind complementcomponent C5. The selection cycle was repeated for six rounds. After thesixth round, the DNA pool representing the selected polypeptides wascloned and sequenced, and the amino acid sequences of candidate C5inhibitors were determined based on the DNA sequences. The polypeptidesequences identified are listed in Table 3.

As used in all of the following tables as well as in the sequencelisting, abbreviations have the following meaning: “Ac” and “NH2”indicate acetyl and amidated termini, respectively; “Nvl” stands fornorvaline; “Phg” stands for phenylglycine; “Sar” stands for sarcosine;“Tbg” stands for tert-butylglycine; “Trt” stands for trityl ortriphenylmethyl; “Chg” stands for cyclohexylglycine; “(N-Me)X” standsfor the N-methylated form of the amino acid indicated by the letter orthree letter abbreviation for that amino acid in place of variable “X”written as N-methyl-X [e.g. (N-Me)A and (N-Me)Ala both stand for theN-methylated form of alanine or N-methyl-alanine]; 7-aza-tryptophan isincorporated where “azaTrp” is indicated; “(4-F)Phe” stands for4-fluorophenylalanine; “Tyr(OMe)” stands for O-methyl tyrosine, “Aib”stands for amino isobutyric acid; “(homo)F” or “(homo)Phe” stands forhomophenylalanine; “(2-OMe)Phg” refers to 2-O-methylphenylglycine;“PropargylGly” refers to propargyl-glycine; “(5-F)W” or “(5-F)Trp”refers to 5-fluorotryptophan; “D-X” refers to the D-stereoisomer of thegiven amino acid “X” wherein the amino acid may be abbreviated usingsingle or three letter code [e.g. (D-Chg) stands for D-cyclohexylglycineand (D-W) stands for D-tryptophan]; “(5-MeO)W” or “(5-MeO)Trp” refers to5-methyl-O-tryptophan; “homoC” refers to homocysteine; “(1-Me-W)” or“(1-Me)W” or “(1-Me-Trp)” or “(1-Me)Trp” refers to 1-methyltryptophan;“Nle” refers to norleucine; 1,2,3,4-tetrahydroisoquinoline-1-carboxylicacid is incorporated where “Tiq” is indicated; “Asp(T)” refers to(S)-2-amino-3-(1H-tetrazol-5-yl)propanoic acid; “(3-C1-Phe)” refers to3-chlorophenylalanine; “[(N-Me-4-F)Phe]” or “(N-Me-4-F)Phe” refers toN-methyl-4-fluorophenylalanine; “Boc” is a tert-Butyloxycarbonylprotecting group; “[xXylyl(y, z)]” refers to the xylyl bridging moietybetween two cysteines where x may be m, p or o to indicate the use ofmeta-, para- or ortho-dibromoxylenes (respectively) to generate bridgingmoieties and the numerical identifiers, y and z, place the amino acidposition within the polypeptide of the cysteines participating in thecyclization; “[cyclo(y,z)]” refers to the formation of a bond betweentwo residues where the numerical identifiers, y and z, place theposition of the residues participating in the bond; “[mXylyl-bicyclo]”indicates that the polypeptide comprises two cyclic loops and that thebridging moiety is generated by reaction with a meta-dibromoxylene. Allother symbols refer to the standard one-letter amino acid code.Additionally, polypeptides comprising PEG2000 or BODIPY-TMR-X sequencetags are indicated.

TABLE 3 Polypeptide Sequences SEQ Compound ID Number Sequence NO R3000Ac-Nvl-C-Y-K-N-Y-H-azaTrp-E-Y-P-Tbg-Y-NH2 1 R3001Ac-Nvl-C-Y-E-N-Tbg-Y-azaTrp-E-Y-(N-Me)G-Nvl-(N-Me)S- 2 NH2 R3002Ac-Nvl-C-Y-E-N-Tbg-Y-azaTrp-E-Y-P-Phg-Nvl-NH2 3 R3003Ac-Nvl-C-Y-E-N-Tbg-Y-azaTrp-E-Y-P-Phg-P-NH2 4 R3004Ac-Nvl-C-Y-N-N-Tbg-E-azaTrp-E-Y-P-Phg-Tbg-NH2 5 R3005Ac-Nvl-C-Y-azaTrp-(N-Me)G-Tbg-Nvl-azaTrp-E-Y-P-Phg-P-NH2 6 R3006Ac-Y-E-N-Tbg-Y-azaTrp-E-Y-(N-Me)G-Nvl-(N-Me)S-NH2 7 R3007[mXylyl(2,7)]Ac-Nvl-C-K-E-Phg-Y-C-(N-Me)S-Tbg-K-azaTrp-E- 8 Y-NH2 R3008[mXylyl(2,10)]Ac-Nvl-C-Phg-T-azaTrp-E-Y-(N-Me)S-H-C-Nvl-P- 9 Nvl-NH2R3020 [mXylyl(2,7)]M-C-S-E-R-Y-C-E-V-R-W-E-Y-NH2 10 R3021[mXylyl(2,7)]M-C-V-E-R-F-C-D-V-Y-W-E-F-NH2 11

Example 4. Polypeptide Synthesis

Polypeptides are synthesized using standard solid-phase Fmoc/tBumethods. The synthesis is typically performed on a Liberty automatedmicrowave peptide synthesizer (CEM, Matthews N.C.) using standardprotocols with Rink amide resin, although other automated synthesizerswithout microwave capability may also be used. All amino acids areobtained from commercial sources unless otherwise noted. The couplingreagent used is2-(6-chloro-1-H-benzotriazole-1yl)-1,1,3,3,-tetramethylaminiumhexafluorophosphate (HCTU) and the base is diisopropylethylamine (DIEA).Polypeptides are cleaved from resin with 95% TFA, 2.5% TIS and 2.5%water for 3 hours and isolated by precipitation with ether. The crudepolypeptides are purified on a reverse phase preparative HPLC using aC18 column, with an acetonitrile/water 0.1% TFA gradient from 20%-50%over 30 min. Fractions containing the pure polypeptide are collected andlyophilized and all polypeptides are analyzed by LC-MS.

Example 5. Formation of Disulfide Cyclized Polypeptides

To produce disulfide cyclized polypeptides, the linear polypeptide isdissolved in a mixture of water and DMSO and the resulting solution isstirred vigorously under an air atmosphere for 12 hrs.

Example 6. Dibromoxylene Polypeptide Cyclization

A 100 mL flask is charged with acetonitrile (12 mL) and water (24 mL)and is degassed with argon for about 5 min. Linear polypeptide (0.01mmole) and 200 mM ammonium bicarbonate (6 mL) are added followed by0.012 mmole or 1,3-bis(bromomethyl) benzene,1,2-bis(bromomethyl)benzene, 1,4-bis(bromomethyl)benzene,2,6-bis(bromomethyl)pyridine or (E)-1,4-dibromobut-2-ene. The reactionmixture is stirred under argon at room temperature for approximately 2hours and monitored by LC-MS. After the reaction is complete, thereaction solution is frozen and lyophilized. HPLC purification of thecrude lyophilized product followed by lyophilization of fractionscontaining pure polypeptide yield the final cyclized product as a whitepowder.

Example 7. Lactam Polypeptide Cyclization

Cyclization of polypeptides using a lactam moiety was performed in thesolid phase. A polypeptide was first synthesized on a solid support Wangresin by standard Fmoc chemistry. Fmoc-ASP(allyl)-OH andFmoc-LYS(alloc)-OH were incorporated in the polypeptide at the indicatedpositions as the two precursor monomers for the lactam bridge formation.After full elongation the resin was washed with dry dichloromethane (3×)and purged with dry Nitrogen gas for 10 min. To remove the allyl andalloc protecting groups, the resin was treated with a 5 fold molarexcess of phenylsilane and purged with Nitrogen for 10 min. A catalyticamount of tetrakis Pd(0) was dissolved in dry dichloromethane and addedto the suspension of resin. After one hour the resin was washedsequentially with dichloromethane (3×), dimethylformamide (3×), sodiumdiethyldithiocarbamate trihydrate (3×), dimethylformamide (3×) anddichloromethane (3×). Lactam cyclization was achieved indimethylformamide (DMF) by treating the deprotected polypeptidecontaining resin with PyAOP((3-Hydroxy-3H-1,2,3-triazolo[4,5-b]pyridinato-O)tri-1-pyrrolidinyl-phosphorushexafluorophosphate) and diisopropylethylamine and allowed to reactovernight. The resin was rinsed with DMF and treated with fresh PyAOPand diisopropylethylamine for additional 60 minutes at 45° C. The resinwas rinsed and washed with dimethylformamide five times. The polypeptidewas cleaved and purified as described in example 4.

Example 8. Triazole Polypeptide Cyclization

Cyclization of polypeptides containing an azide and an alkyne moiety wasperformed on the solid phase. Polypeptide containing resin (0.05 mmol)was treated with dichloromethane and allowed to swell for 10 min. Thesolvent was then exchanged to DMF (3-5 mL) and after 10 min, a solutionof Cu-TBTA ligand was added (125 μL of a 20 mM solution). The suspensionwas purged with Argon gas and then ascorbic acid (5 moles) was added.The solution was shaken for 2 h and the excess reagents removed, theresin was washed with a solution of EDTA in DMF to remove excess copper.The polypeptide was cleaved and purified as described in Example 4.

Example 9. Polypeptides of the Current Invention

Polypeptides of the current invention were synthesized. These includethe compounds listed in Table 4.

TABLE 4 Compounds of the invention SEQ Compound ID Number Sequence NO.R3000 Ac-Nvl-C-Y-K-N-Y-H-azaTrp-E-Y-P-Tbg-Y-NH2 1 R3001Ac-Nvl-C-Y-E-N-Tbg-Y-azaTrp-E-Y-(N-Me)G-Nvl-(N-Me)S-NH2 2 R3002Ac-Nvl-C-Y-E-N-Tbg-Y-azaTrp-E-Y-P-Phg-Nvl-NH2 3 R3003Ac-Nvl-C-Y-E-N-Tbg-Y-azaTrp-E-Y-P-Phg-P-NH2 4 R3004Ac-Nvl-C-Y-N-N-Tbg-E-azaTrp-E-Y-P-Phg-Tbg-NH2 5 R3005Ac-Nvl-C-Y-azaTrp-(N-Me)G-Tbg-Nvl-azaTrp-E-Y-P-Phg-P-NH2 6 R3006Ac-Y-E-N-Tbg-Y-azaTrp-E-Y-(N-Me)G-Nvl-(N-Me)S-NH2 7 R3007[mXylyl(2,7)]Ac-Nvl-C-K-E-Phg-Y-C-(N-Me)S-Tbg-K-azaTrp-E-Y- 8 NH2 R3008[mXylyl(2,10)]Ac-Nvl-C-Phg-T-azaTrp-E-Y-(N-Me)S-H-C-Nvl-P- 9 Nvl-NH2R3020 [mXylyl(2,7)]M-C-S-E-R-Y-C-E-V-R-W-E-Y-NH2 10 R3021[mXylyl(2,7)]M-C-V-E-R-F-C-D-V-Y-W-E-F-NH2 11 R3079Nvl-Nvl-Y-E-N-Tbg-Y-azaTrp-E-Y-P-Phg-Nvl-NH2 12 R3055Ac-Nvl-S-Y-E-N-Tbg-Y-azaTrp-E-Y-P-Phg-Nvl-NH2 13 R3120Ac-Nvl-Nvl-Y-E-(N-Me)N-Tbg-Y-azaTrp-E-Y-P-Chg-Nvl-NH2 14 R3057[mXylyl(2,7)]M-C-V-E-R-F-C-D-Tbg-Y-azaTrp-E-Y-P-Phg-Nvl- 15 NH2 R3056Ac-Nvl-Nvl-Y-E-N-Tbg-Y-azaTrp-E-Y-P-Phg-Nvl-NH2 16 R3054Ac-Nvl-C-Y-E-N-Tbg-Y-azaTrp-E-Y-P-Chg-Nvl-NH2 17 R3029Ac-Nvl-C-Y-E-N-Tbg-Y-azaTrp-E-Y-P-Phg-NH2 18 R3048[mXylyl(1,6)]Ac-C-V-E-R-F-C-D-V-Y-W-E-F-NH2 19 R3072Ac-Nvl-Nvl-Y-E-N-Tbg-Y-azaTrp-E-Y-P-Chg-Nvl-K-NH2 20 R3024Ac-C-Y-E-N-Tbg-Y-azaTrp-E-Y-P-Phg-Nvl-NH2 21 R3114Ac-Nvl-Nvl-(N-Me)Y-E-N-Tbg-Y-azaTrp-E-Y-P-Chg-Nvl-NH2 22 R3050[pXylyl(2,10)]Ac-Nvl-C-Phg-T-azaTrp-E-Y-(N-Me)S-H-C-Nvl-NH2 23 R3025Ac-Y-E-N-Tbg-Y-azaTrp-E-Y-P-Phg-Nvl-NH2 24 R3061Ac-Nvl-S-Y-E-A-Tbg-Y-azaTrp-E-Y-P-Chg-Nvl-NH2 25 R3041Ac-Y-E-N-Tbg-Y-W-E-Y-P-Phg-Nvl-NH2 26 R3077Ac-Nvl-Nvl-Y-E-N-Tbg-Y-azaTrp-E-Y-P-Chg-Nvl-K-(PEG2000) 27 NH2 R3030Ac-Nvl-C-Y-E-N-Tbg-Y-azaTrp-E-Y-P-NH2 28 R3062Ac-Nvl-S-Y-E-N-A-Y-azaTrp-E-Y-P-Chg-Nvl-NH2 29 R3066Ac-Nvl-S-Y-E-N-Tbg-A-azaTrp-E-Y-P-Chg-Nvl-NH2 30 R3011[mXylyl(2,10)]Ac-Nvl-C-Phg-T-azaTrp-E-Y-(N-Me)S-H-C-Nvl-P- 31 NH2 R3070Ac-Nvl-S-Y-E-N-Tbg-Y-azaTrp-E-Y-A-Chg-Nvl-NH2 32 R3071Ac-Nvl-S-Y-E-N-Tbg-Y-azaTrp-E-Y-P-A-Nvl-NH2 33 R3033[mXylyl(2,10)]Ac-Nvl-C-Phg-A-azaTrp-E-Y-(N-Me)S-H-C-Nvl-NH2 34 R3038[mXylyl(2,10)]Ac-Nvl-C-Phg-T-azaTrp-E-Y-(N-Me)S-A-C-Nvl-NH2 35 R3012[mXylyl(2,10)]Ac-Nvl-C-Phg-T-azaTrp-E-Y-(N-Me)S-H-C-Nvl-NH2 36 R3060Ac-Nvl-S-Y-A-N-Tbg-Y-azaTrp-E-Y-P-Chg-Nvl-NH2 37 R3039[mXylyl(2,10)]Ac-Nvl-C-Phg-T-azaTrp-E-Y-(N-Me)S-H-C-A-NH2 38 R3037[mXylyl(2,10)]Ac-Nvl-C-Phg-T-azaTrp-E-Y-(N-Me)A-H-C-Nvl-NH2 39 R3076Ac-Nvl-Nvl-Y-E-N-Tbg-Y-azaTrp-E-Y-P-Chg-Nvl-K-(BODIPY- 40 TMR-X)NH2R3074 [mXylyl(2,10)]Ac-Nvl-C-Phg-T-azaTrp-E-Tyr(OMe)-(N-Me)S-H-C- 41Nvl-NH2 R3013 [mXylyl(2,10)]Ac-Nvl-C-Phg-T-azaTrp-E-Y-(N-Me)S-H-C-NH2 42R3065 [pXylyl(2,10)]Ac-Nvl-C-Phg-T-azaTrp-E-Y-P-H-C-Nvl-NH2 43 R3073[mXylyl(2,10)]Ac-Nvl-C-Phg-T-azaTrp-E-Phe(4-F)-(N-Me)S-H-C- 44 Nvl-NH2R3116 Ac-Nvl-Nvl-Y-E-N-Tbg-Y-(N-Me)W-E-Y-P-Chg-Nvl-NH2 45 R3091[mXylyl(2,10)]Ac-Nvl-C-Phg-T-W-E-Y-(N-Me)S-A-C-Nvl-NH2 46 R3078PEG2000-Nvl-Nvl-Y-E-N-Tbg-Y-azaTrp-E-Y-P-Phg-Nvl-NH2 47 R3100[mXylyl(2,10)]Ac-Nvl-C-Phg-T-azaTrp-E-F-(N-Me)S-A-C-Nvl-NH2 48 R3121Ac-Nvl-Nvl-Y-E-N-Tbg-Y-azaTrp-E-Y-P-(N-Me)Phg-Nvl-NH2 49 R3043[mXylyl(2,7)]M-C-V-E-R-F-C-D-V-Y-W-E-NH2 50 R3102[mXylyl(2,10)]Ac-Nvl-C-Phg-T-azaTrp-E-Y-P-H-C-Nvl-NH2 51 R3026Ac-E-N-Tbg-Y-azaTrp-E-Y-P-Phg-Nvl-NH2 52 R3031[mXylyl(2,10)]Ac-A-C-Phg-T-azaTrp-E-Y-(N-Me)S-H-C-Nvl-NH2 53 R3019[mXylyl(2,14)]Ac-Nvl-C-Y-E-N-Tbg-Y-azaTrp-E-Y-P-Phg-Nvl-C- 54 NH2 R3014[mXylyl(1,9)]Ac-C-Phg-T-azaTrp-E-Y-(N-Me)S-H-C-Nvl-P-Nvl- 55 NH2 R3104[pXylyl(2,10)]Ac-Nvl-homoC-Phg-T-azaTrp-E-Y-(N-Me)S-H-C-Nvl- 56 NH2R3059 Ac-Nvl-S-A-E-N-Tbg-Y-azaTrp-E-Y-P-Chg-Nvl-NH2 57 R3115Ac-Nvl-Nvl-Y-(N-Me)E-N-Tbg-Y-azaTrp-E-Y-P-Chg-Nvl-NH2 58 R3110Ac-Y-E-N-Tbg-Y-(1-Me)W-E-Y-P-Phg-Nvl-NH2 59 R3126Ac-Nvl-C-Y-N-N-Tbg-E-azaTrp-E-C-P-Phg-Tbg-NH2 60 R3049[oXylyl(2,10)]Ac-Nvl-C-Phg-T-azaTrp-E-Y-(N-Me)S-H-C-Nvl-NH2 61 R3069Ac-Nvl-S-Y-E-N-Tbg-Y-azaTrp-E-A-P-Chg-Nvl-NH2 62 R3015[mXylyl(1,9)]Ac-C-Phg-T-azaTrp-E-Y-(N-Me)S-H-C-NH2 63 R3068Ac-Nvl-S-Y-E-N-Tbg-Y-azaTrp-A-Y-P-Chg-Nvl-NH2 64 R3105[mXylyl(2,10)]Ac-Nvl-C-Phg-T-azaTrp-E-Y-(N-Me)S-H-homoC- 65 Nvl-NH2R3106 [pXylyl(2,10)]Ac-Nvl-C-Phg-T-azaTrp-E-Y-(N-Me)S-H-homoC-Nvl- 66NH2 R3111 [mXylyl(4,10)]Ac-Nvl-T-Phg-C-azaTrp-E-Y-(N-Me)S-A-C-Nvl-NH2 67R3112 [mXylyl(2,10)]Ac-Nle-C-Phg-T-azaTrp-E-Y-(N-Me)S-H-C-Nvl-NH2 68R3113 [mXylyl(3,11)]Ac-Y-Nvl-C-Phg-T-azaTrp-E-Y-(N-Me)S-H-C-Nvl- 69 NH2R3134 [mXylyl(2,10)]Ac-Nvl-C-Phg-T-azaTrp-E-(3-Cl-Phe)-(N-Me)S-A-C- 70Nvl-NH2 R3018[mXylyl(2,10)]Ac-Nvl-C-Y-E-N-Tbg-Y-azaTrp-E-C-P-Phg-Nvl-NH2 71 R3027Ac-N-Tbg-Y-azaTrp-E-Y-P-Phg-Nvl-NH2 72 R3028Ac-Tbg-Y-azaTrp-E-Y-P-Phg-Nvl-NH2 73 R3032[mXylyl(2,10)]Ac-Nvl-C-A-T-azaTrp-E-Y-(N-Me)S-H-C-Nvl-NH2 74 R3058[pXylyl(2,10)]Ac-Nvl-C-Chg-T-azaTrp-E-Y-(N-Me)S-H-C-Nvl-NH2 75 R3067Ac-Nvl-S-Y-E-N-Tbg-Y-A-E-Y-P-Chg-Nvl-NH2 76 R3117Ac-Nvl-Nvl-Y-E-N-Tbg-Y-azaTrp-E-(N-Me)Y-P-Chg-Nvl-NH2 77 R3022Ac-Nvl-C-Phg-T-azaTrp-E-Y-(N-Me)S-H-C-Nvl-P-Nvl-NH2 78 R3016[mXylyl(1,9)]Ac-C-Tbg-Y-azaTrp-E-Y-(N-Me)S-H-C-NH2 79 R3089[mXylyl(2,10)]Ac-Chg-C-Phg-T-azaTrp-E-Y-(N-Me)S-A-C-Nvl-NH2 80 R3083[mXylyl(2,10)]Ac-V-C-Phg-T-azaTrp-E-Y-(N-Me)S-A-C-Nvl-NH2 81 R3087[mXylyl(2,10)]Ac-Nvl-C-(2-0Me)Phg-T-azaTrp-E-Y-(N-Me)S-H-C- 82 Nvl-NH2R3103 [mXylyl(2,10)]Ac-Nvl-homoC-Phg-T-azaTrp-E-Y-(N-Me)S-H-C- 83Nvl-NH2 R3135 [mXylyl(2,10)]Ac-Nvl-C-Phg-T-azaTrp-E-Y-(N-Me)S-(D-Ala)-C-84 Nvl-NH2 R3034 [mXylyl(2,10)]Ac-Nvl-C-Phg-T-A-E-Y-(N-Me)S-H-C-Nvl-NH285 R3035 [mXylyl(2,10)]Ac-Nvl-C-Phg-T-azaTrp-A-Y-(N-Me)S-H-C-Nvl-NH2 86R3036 [mXylyl(2,10)]Ac-Nvl-C-Phg-T-azaTrp-E-A-(N-Me)S-H-C-Nvl-NH2 87R3044 [mXylyl(2,7)]M-C-V-E-R-F-C-D-V-Y-W-NH2 88 R3080[mXylyl(2,9)]Ac-Nvl-C-Phg-T-azaTrp-E-Y-(N-Me)S-C-Nvl-NH2 89 R3085[mXylyl(2,10)]heptanoyl-Nvl-C-Phg-T-azaTrp-E-Y-(N-Me)S-A-C- 90 Nvl-NH2R3086 [mXylyl(5,13)]Ac-Nvl-S-Y-E-C-Tbg-Y-azaTrp-E-Y-P-Chg-C-Nvl- 91 NH2R3092 [mXylyl(2,10)]Ac-Nvl-C-Phg-T-F-E-Y-(N-Me)S-A-C-Nvl-NH2 92 R3095[mXylyl(2,10)]Ac-Nvl-C-Phg-T-azaTrp-E-(homo)F-(N-Me)S-A-C- 93 Nvl-NH2R3096 [mXylyl(2,10)]Ac-Nvl-C-Phg-Aib-azaTrp-E-Y-(N-Me)S-H-C-Nvl- 94 NH2R3122 [mXylyl(2,10)]Ac-Nvl-C-Tiq-T-azaTrp-E-Y-(N-Me)S-H-C-Nvl-NH2 95R3075 [mXylyl(2,11)]Nvl-C-Y-(N-Me)S-Phg-(N-Me-4-F)Phe-(N-Me)S-H- 96(N-Me-4-F)Phe-G-C-NH2 R3107[mXylyl(2,10)]Ac-Nvl-homoC-Phg-T-azaTrp-E-Y-(N-Me)S-H- 97 homoC-Nvl-NH2R3108 [pXylyl(2,10)]Ac-Nvl-homoC-Phg-T-azaTrp-E-Y-(N-Me)S-H- 98homoC-Nvl-NH2 R3127[mXylyl(2,10)]Ac-Nvl-C-Y-N-N-Tbg-E-azaTrp-E-C-P-Phg-Tbg-NH2 99 R3133[mXylyl(2,10)]Ac-Nvl-C-Phg-(D-Ala)-azaTrp-E-Y-(N-Me)S-H-C- 100 Nvl-NH2R3009 [mXylyl(2,10)]Ac-Nvl-C-Y-E-(N-Me)G-Tbg-Y-azaTrp-E-C-Nvl-P- 101Nvl-NH2 R3010[mXylyl(2,13)]Ac-Nvl-C-Y-E-(N-Me)G-Tbg-Y-azaTrp-E-Nvl-Nvl-P- 102 C-NH2R3017 [mXylyl(2,8)]Ac-Nvl-C-Y-E-N-Tbg-Y-C-E-Y-P-Phg-Nvl-NH2 103 R3023Ac-Y-P-Y-C-Phg-azaTrp-Tbg-E-Nvl-N-Y-Nvl-E-NH2 104 R3040[cyclo(2,10)]Ac-Nvl-C-Phg-T-azaTrp-E-Y-(N-Me)S-H-C-Nvl-P-Nvl 105 R3042[cyc1o(2,10)]Ac-Nvl-C-Phg-T-azaTrp-E-Y-(N-Me)S-H-C-Nvl-NH2 106 R3045[mXylyl(2,7)]M-C-V-E-R-F-C-D-V-Y-NH2 107 R3046[mXylyl(2,7)]M-C-V-E-R-F-C-D-V-NH2 108 R3047[mXylyl(2,7)]M-C-V-E-R-F-C-NH2 109 R3051[mXylyl(2,11)]Nvl-C-Y-(N-Me)S-Phg-(N-Me-4-F)Phe-(N-Me)S-H- 110(N-Me-4-F)Phe-(N-Me)G-C-NH2 R3052[mXylyl(2,9)]Nvl-C-Y-Tbg-Phg-N-(N-Me)G-L-C-Phg-(N-Me)A- 111 NH2 R3053[mXylyl-bicyclo]Nvl-C-C-N-Tbg-Phg-C-Tbg-(N-Me)S-C-Tbg-NH2 112 R3063Ac-Tbg-Y-azaTrp-E-Y-NH2 113 R3064 Ac-Y-azaTrp-E-Y-P-NH2 114 R3081Ac-Y-E-N-Tbg-Y-azaTrp-(N-Me)E-Y-P-Phg-Nvl-NH2 115 R3082[mXylyl(1,9)]heptanoyl-C-Phg-T-azaTrp-E-Y-(N-Me)S-A-C-Nvl- 116 NH2 R3084[mXylyl(2,10)]Ac-Nvl-C-Phg-T-azaTrp-E-Y-S-A-C-Nvl-NH2 117 R3088[mXylyl(2,10)]Ac-Nvl-C-Phg-T-(5-F)W-E-Y-(N-Me)S-A-C-Nvl- 118 NH2 R3090[mXylyl(2,10)]Ac-Nvl-C-F-T-azaTrp-E-Y-(N-Me)S-A-C-Nvl-NH2 119 R3093[mXylyl(2,10)]Ac-Nvl-C-(D-Chg)-T-azaTrp-E-Y-(N-Me)S-A-C-Nvl- 120 NH2R3094 Ac-Y-E-N-Tbg-Y-(5-Me0)W-E-Y-P-Phg-Nvl-NH2 121 R3097[mXylyl(2,10)]Ac-Nvl-C-Phg-T-azaTrp-D-Y-(N-Me)S-A-C-Nvl-NH2 122 R3098[mXylyl(2,10)]Ac-Nvl-C-Phg-T-azaTrp-Q-Y-(N-Me)S-A-C-Nvl-NH2 123 R3099[mXylyl(2,10)]Ac-Nvl-C-Phg-T-azaTrp-N-Y-(N-Me)S-A-C-Nvl-NH2 124 R3101[mXylyl(2,10)]Ac-Nvl-C-Phg-T-azaTrp-E-Y-(N-Me)S-H-G-C-Nvl- 125 NH2 R3109[mXylyl(2,10)]Ac-Nvl-C-Phg-T-(1-Me-W)-E-Y-(N-Me)S-A-C-Nvl- 126 NH2 R3118Ac-Y-E-N-Tbg-Y-(D-Trp)-E-Y-P-Phg-Nvl-NH2 127 R3119Ac-Y-E-N-Y-(D-Trp)-E-Y-P-Phg-Nvl-NH2 128 R3123Ac-Y-E-N-Tbg-Y-azaTrp-(D-G1u)-Y-P-Phg-Nvl-NH2 129 R3124[mXylyl(1,6)]Ac-C-V-E-R-F-C-V-Y-W-E-F-NH2 130 R3125[mXylyl(1,6)]Ac-C-V-E-R-F-C-W-E-F-NH2 131 R3128Ac-Nvl-C-Y-N-N-Tbg-E-C-E-Y-P-Phg-Tbg-NH2 132 R3129[mXylyl(2,8)]Ac-Nvl-C-Y-N-N-Tbg-E-C-E-Y-P-Phg-Tbg-NH2 133 R3130Ac-Nvl-Nvl-Y-E-N-Tbg-(N-Me)Y-azaTrp-E-Y-P-Chg-Nvl-NH2 134 R3131[mXylyl(2,10)]Ac-Nvl-C-Phg-T-W-Asp (T)-Y-(N-Me)S-H-C-Nvl- 135 NH2 R3132[mXylyl(2,10)]Ac-Nvl-C-Phg-T-(D-Trp)-E-Y-(N-Me)S-H-C-Nvl- 136 NH2 R3136[mXylyl(2,10)]heptanoyl-Nvl-C-(D-Phg)-T-azaTrp-E-Y-(N-Me)S-A- 137C-Nvl-NH2 R3137[mXylyl(1,9)]heptanoyl-C-(D-Phg)-T-azaTrp-E-Y-(N-Me)S-A-C- 138 Nvl-NH2R3138 [mXylyl(1,6)]Ac-C-V-E-R-F-C-D-Tbg-Y-W-E-F-NH2 139 R3139[mXylyl(1,6)]Ac-C-Tbg-E-R-F-C-D-Tbg-Y-W-E-F-NH2 140 R3140[mXylyl(1,6)]Ac-C-V-E-R-F-C-D-V-Y-W-E-Y-P-NH2 141 R3141[mXylyl(1,6)]Ac-C-V-E-R-F-C-D-V-Y-W-E-F-P-NH2 142 R3142[mXylyl(1,6)]Ac-C-V-E-R-F-C-D-V-Y-azaTrp-E-Y-P-NH2 143 R3143[mXylyl(1,6)]Ac-C-V-E-R-F-C-D-Tbg-Y-W-E-Y-P-NH2 144 R3144[mXylyl(1,6)]Ac-C-V-E-R-F-C-D-Tbg-Y-azaTrp-E-Y-P-Phg-Nvl- 145 NH2 R3145[mXylyl(1,6)]Ac-C-V-E-R-F-C-D-Tbg-Y-azaTrp-E-Y-P-(D-Phg)- 146 Nvl-NH2R3146 [mXylyl(1,6)]Ac-C-Tbg-E-R-F-C-D-V-Y-W-E-F-NH2 147 R3147[mXylyl(1,6)]Ac-C-V-E-R-F-C-D-V-Y-W-E-F-Propargyl-Gly-NH2 148 R3148[mXylyl(1,6)]Ac-C-A-E-R-F-C-D-Tbg-Y-W-E-Y-P-Phg-Nvl-NH2 149 R3149[mXylyl(1,6)]Ac-C-A-E-R-F-C-D-Tbg-Y-W-E-Y-P-(D-Phg)-Nvl- 150 NH2 R3150[mXylyl(1,6)]Ac-C-V-A-R-F-C-D-Tbg-Y-azaTrp-E-Y-P-Phg-Nvl- 151 NH2 R3151[mXylyl(1,6)]Ac-C-V-A-R-F-C-D-Tbg-Y-azaTrp-E-Y-P-(D-Phg)- 152 Nvl-NH2R3152 [mXylyl(1,6)]Ac-C-V-E-R-F-C-D-Tbg-Y-azaTrp-E-Y-P-Chg-Nvl- 153 NH2R3153 [mXylyl(1,6)]Ac-C-V-E-A-F-C-D-Tbg-Y-azaTrp-E-Y-P-Phg-Nvl- 154 NH2R3154 [mXylyl(1,6)]Ac-C-V-E-A-F-C-D-Tbg-Y-azaTrp-E-Y-P-(D-Phg)- 155Nvl-NH2 R3155 [mXylyl(1,6)]Ac-C-V-E-R-A-C-D-Tbg-Y-azaTrp-E-Y-P-Phg-Nvl-156 NH2 R3156 [mXylyl(1,6)]Ac-C-V-E-R-A-C-D-Tbg-Y-azaTrp-E-Y-P-(D-Phg)-157 Nvl-NH2 R3157[mXylyl(1,6)]Ac-C-V-E-R-F-C-A-Tbg-Y-azaTrp-E-Y-P-Phg-Nvl- 158 NH2 R3158[mXylyl(1,6)]Ac-C-V-E-R-F-C-A-Tbg-Y-azaTrp-E-Y-P-(D-Phg)- 159 Nvl-NH2R3159 [mXylyl(1,6)](des-amino)C-V-E-R-F-C-D-Tbg-Y-azaTrp-E-Y-P-Phg- 160Nvl-NH2 R3160[mXylyl(1,6)](des-amino)C-V-E-R-F-C-D-Tbg-Y-azaTrp-E-Y-P-(D- 161Phg)-Nvl-NH2 R3161[mXylyl(1,6)]Ac-C-A-E-R-F-C-D-Tbg-Y-azaTrp-E-Y-P-Phg-K-NH2 162 R3162[mXylyl(1,6)]Ac-C-A-E-R-F-C-D-Tbg-Y-azaTrp-E-Y-P-(D-Phg)-K- 163 NH2R3163 [cyclo(1,6)]Ac-C-V-E-R-F-C-D-Tbg-Y-azaTrp-E-Y-P-Phg-Nvl-NH2 164R3164 [mXylyl(1,6)]Ac-C-A-E-R-F-C-D-Tbg-Y-azaTrp-E-Y-P-Phg-(Lys- 165C12)-NH2 R3165[mXylyl(1,6)]Ac-C-A-E-R-F-C-D-Tbg-Y-azaTrp-E-Y-P-Phg-(Lys- 166 C10)-NH2R3166 [mXylyl(1,6)]Ac-C-A-E-R-F-C-D-Tbg-Y-azaTrp-E-Y-P-Phg-(Lys- 167C8)-NH2 R3167[mXylyl(1,6)]Ac-C-V-E-R-F-C-(alpha-methyl)D-Tbg-Y-azaTrp-E-Y- 168P-Chg-Nvl-NH2 R3168[mXylyl(1,6)]Ac-C-V-E-R-F-C-Asp(T)-Tbg-Y-azaTrp-E-Y-P-Chg- 169 Nvl-NH2R3169 [cyclo(1,6)]Ac-K-V-E-R-F-D-D-Tbg-Y-azaTrp-E-Y-P-Chg-Nvl-NH2 170R3170 [mXylyl(1,6)]Ac-C-A-E-R-F-C-D-Tbg-Y-azaTrp-E-Y-P-Phg-K 171 R3171[mXylyl(1,6)]Ac-C-A-E-R-F-C-D-Tbg-Y-azaTrp-E-Y-P-Phg-(Lys- 172 C12)R3172 [mXylyl(1,6)]Ac-C-V-E-R-F-C-(N-Me)D-Tbg-Y-azaTrp-E-Y-P-Chg- 173Nvl-NH2 R3173 [cyclo(1,6)]Ac-K-V-E-R-F-D-D-Tbg-Y-azaTrp-E-Y-P-Chg-Nvl174 R3174 [cyclo(1,6)]Ac-K-V-E-R-F-D-Asp(T)-Tbg-Y-azaTrp-E-Y-P-Chg-Nvl175 R3175 [cyclo(1,6)]Ac-K-V-E-R-F-D-D-Tbg-Y-azaTrp-E-Y-P-Chg-B20 176R3176 [cyclo(1,6)]Ac-K-V-E-R-F-D-(N-Me)D-Tbg-Y-azaTrp-E-Y-P-Chg- 177 NvlR3177 [mXylyl(1,6)]Ac-C-V-E-R-F-C-D-Tbg-Y-azaTrp-E-W-P-Chg-Nvl 178 R3178[mXylyl(1,6)]Ac-C-V-E-R-F-C-D-Tbg-Y-azaTrp-E-(homo)Phe-P- 179 Chg-NvlR3179 [mXylyl(1,6)]Ac-C-V-E-R-F-C-D-Tbg-Y-azaTrp-E-(m-C1-homo)Phe- 180P-Chg-Nvl R3180[mXylyl(1,6)]Ac-C-V-E-R-F-C-D-Tbg-Y-azaTrp-E-2Nal-P-Chg-Nvl 181 R3181[mXylyl(1,6)]Ac-C-V-E-R-F-C-D-Tbg-Y-(3-aminomethyl)Phe-E-Y- 182P-Chg-Nvl R3182[cyclo-triazolyl(1,6)]Ac-X02-V-E-R-F-X31-D-Tbg-Y-azaTrp-E-Y-P- 183Chg-Nvl R3183 [cyclo(1,6)]Ac-K-V-E-R-F-D-(N-Me)D-Tbg-Y-azaTrp-E-Y-P-Chg-184 (Lys-C16) R3184[cyclo-thioalkyl(1,5)]V-E-R-F-C-D-Tbg-Y-azaTrp-E-Y-P-Chg-Nvl 185 R3185[mXylyl(1,6)]Ac-C-V-E-R-F-C-C1e-Tbg-Y-azaTrp-E-Y-P-Chg-Nvl 186 R3186[mXylyl(1,6)]Ac-C-V-E-R-F-C-(Ac-Pyran)-Tbg-Y-azaTrp-E-Y-P- 187 Chg-NvlR3187 [mXylyl(1,6)]Ac-C-V-E-R-F-C-D-Tbg-Y-azaTrp-E-(3- 188aminomethyl)Phe-P-Chg-Nvl R3188[cyclo-olefinyl(1,6)]Ac-X30-V-E-R-F-X12-D-Tbg-Y-azaTrp-E-Y-P- 189Chg-Nvl R3189 [mXylyl(1,6)]Ac-C-A-E-R-F-C-D-Tbg-Y-azaTrp-E-Y-P-Phg-(Lys-190 C16) R3190[cyclo(1,6)]Ac-K-V-E-R-F-D-(N-Me)D-Tbg-Y-azaTrp-E-Y-P-Chg- 191 B20 R3191[cyclo(1,6)]Ac-K-V-E-R-F-D-(N-Me)D-Tbg-Y-azaTrp-E-Y-P-Chg-K 192 R3192[cyclo(1,6)]Ac-K-V-E-R-F-D-(N-Me)D-Tbg-Y-azaTrp-E-Y-P-Chg- 193 K-NH2R3193 [cyclo(1,6)]Ac-K-V-E-R-F-D-(N-Me)D-Tbg-Y-azaTrp-E-Y-P-Chg- 194 B28R3194 [cyclo(1,6)]Ac-K-V-E-R-F-D-(N-Me)D-Tbg-Y-azaTrp-E-Y-P-Chg- 195(Lys-C16)-NH2 R3195[cyclo(1,6)]Ac-K-V-E-R-F-D-(N-Me)D-Tbg-Y-W-E-Y-P-Chg-(Lys- 196 C16)R3196 [cyclo(1,6)]Ac-K-V-E-R-F-D-(N-Me)D-Tbg-Y-W-E-Y-P-Chg-K 197 R3197[cyclo(1,6)]Ac-K-V-E-R-F-D-(N-Me)D-Tbg-Y-W-E-Y-P-Chg-K14 198 R3198[cyclo(1,6)](desamino)C-V-E-R-F-C-(N-Me)D-Tbg-Y-azaTrp-E-Y-P- 199Chg-(Lys-C16) R3199[cyclo(1,6)](desamino)C-(D-Ala)-E-R-F-C-(N-Me)D-Tbg-Y-azaTrp- 200E-Y-P-Chg-(Lys-C16) R3200[cyclo(1,6)]Ac-K-V-E-R-F-D-(N-Me)D-Tbg-Y-azaTrp-E-Y-P-Aib- 201 (Lys-C16)

Polypeptides R3183 (SEQ ID NO: 184) and R3193 (SEQ ID NO: 194) weresynthesized according to the amino acid sequences of R3176 (SEQ ID NO:177) with the exception of the replacement of Nvl-NH2 with Lys. The sidechain amine group of the Lys residue was modified with the differentlipophilic moieties resulting in lipidated polypeptides.

Example 10. Optimization and Testing of C5 Inhibitors

Polypeptides selected according to Example 3 and listed in Table 3 weretested for their ability to inhibit complement-mediated cell lysis.Additionally, a variety of optimized polypeptides were synthesizedaccording to the methods of Examples 4-8 and tested as well (see Table4). Optimized polypeptide sequences include those obtained by making avariety of truncations, deletions, additions and/or substitutions tocompounds R3002 (SEQ ID NO: 3), R3008 (SEQ ID NO: 9) and R3021 (SEQ IDNO: 11) or through the formation of hybrid polypeptides comprisingcombinations of regions selected from any of the three.

Human Hemolysis Assay (RBC Lysis Assay Using Complete Human Sera)

Polypeptides listed in Table 3, as well as their optimized derivatives(see Table 4) were assessed for inhibitor activity using a red bloodcell hemolysis assay. Antibody-sensitized sheep erythrocytes (ComplementTechnology, Tyler, Tex.) were plated at 2.5×10⁷ cells/well with completehuman sera (Complement Technology, Tyler Tex.) and polypeptides todetermine the inhibitory effect of the polypeptides on the lysis of redblood cells. Cells were centrifuged for 3 minutes at 2,090×gravity andresuspended in fresh GVB++ buffer (Complement Technology, Tyler Tex.).Human sera was rapidly thawed at 37° C. and then stored on ice untildiluted into GVB++. Ten 6-fold serial dilutions of polypeptides (10 mMstock, DMSO) were performed in DMSO and then added to buffer. 50 μl ofeach polypeptide dilution was combined with sera and 100 μl of cells inindividual wells of a 96-well tissue culture-treated clear microtitreplate (USA Scientific, Ocala, Fla.) and resuspended by pipetting.Samples were incubated at 37° C. for one hour. Following incubation,plates were centrifuged at 2,090×gravity for 2 minutes. 100 μl ofsupernatant was transferred to a new plate and the absorbance was readat 412 nm. Data was fit with a log-logit formula producing adose-response curve and IC₅₀. As used herein, the term “IC₅₀” refers tothe half maximal inhibitory concentration, a value used to indicate theamount of the inhibitor needed to reduce a given reaction or process byhalf. Compounds tested are listed in Table 5.

TABLE 5 Compounds analyzed Compound Avg. SEQ ID Number IC₅₀ (nM) NO.R3000 >10,000 1 R3001 67.2 2 R3002 11.9 3 R3003 13.9 4 R3004 53.5 5R3005 66.7 6 R3006 267 7 R3007 314 8 R3008 97 9 R3009 >100,000 101R3010 >100,000 102 R3011 112 31 R3012 148.5 36 R3013 344 42 R3014 142055 R3015 >3,960 63 R3016 >13,000 79 R3017 >100,000 103 R3018 >5,730 71R3019 1320 54 R3020 24.6 10 R3021 27.5 11 R3022 >10,200 78R3023 >100,000 104 R3024 32.5 21 R3025 47.5 24 R3026 1020 52R3027 >10,000 72 R3028 >10,000 73 R3029 18.5 18 R3030 83.6 28 R3031 109053 R3032 >10,000 74 R3033 131 34 R3034 >50,000 85 R3035 >50,000 86R3036 >50,000 87 R3037 276 39 R3038 140 35 R3039 240 38 R3040 >100,000105 R3041 71.3 26 R3042 >100,000 106 R3043 934 50 R3044 >50,000 88R3045 >100,000 107 R3046 >100,000 108 R3047 >100,000 109 R3048 19.3 19R3049 >3,100 61 R3050 42.9 23 R3051 >100,000 110 R3052 >100,000 111R3053 >100,000 112 R3054 14.1 17 R3055 10.4 13 R3056 13.8 16 R3057 12.415 R3058 >10,000 75 R3059 2160 57 R3060 161 37 R3061 53.9 25 R3062 89.929 R3063 >100,000 113 R3064 >100,000 114 R3065 394 43 R3066 104 30R3067 >10,000 76 R3068 >4,500 64 R3069 >3,670 62 R3070 123 32 R3071 12833 R3072 26.9 20 R3073 403 44 R3074 308 41 R3075 >75,000 96 R3076 297 40R3077 81.7 27 R3078 568 47 R3079 7.3 12 R3080 >50,000 89 R3081 >100,000115 R3083 >25,000 81 R3084 >100,000 117 R3086 >50,000 91 R3087 >25,00082 R3088 >100,000 118 R3089 >15,000 80 R3090 >100,000 119 R3091 483 46R3092 >50,000 92 R3093 >100,000 120 R3094 >100,000 121 R3095 >50,000 93R3096 >50,000 94 R3097 >100,000 122 R3098 >100,000 123 R3099 >100,000124 R3100 626 48 R3101 >100,000 125 R3102 978 51 R3103 >25,000 83R3104 >2,000 56 R3105 >5,000 65 R3106 >5,000 66 R3107 >75,000 97R3108 >75,000 98 R3109 >100,000 126 R3110 2940 59 R3111 >5,000 67R3112 >5,000 68 R3113 >5,000 69 R3114 36.6 22 R3115 2780 58 R3116 441 45R3117 >10,000 77 R3118 >100,000 127 R3119 >100,000 128 R3120 12.2 14R3121 804 49 R3122 >50,000 95 R3123 >100,000 129 R3124 >100,000 130R3125 >100,000 131 R3126 >3000 60 R3127 >75,000 99 R3128 >100,000 132R3129 >100,000 133 R3130 >100,000 134 R3131 >100,000 135 R3132 >100,000136 R3133 >75,000 100 R3134 >5,000 70 R3135 >25,000 84 R3136 >50,000 137R3137 >100,000 138 R3138 87.2 139 R3139 97.2 140 R3140 17.9 141 R314124.5 142 R3142 44.6 143 R3143 18.6 144 R3144 6.7 145 R3145 39 146 R3146107 147 R3147 138 148 R3148 8.5 149 R3149 13.6 150 R3150 32 151 R3151165 152 R3152 11 153 R3153 175 154 R3154 592 155 R3155 1530 156R3156 >10,000 157 R3157 84.5 158 R3158 327 159 R3159 7.6 160 R3160 37.1161 R3161 7 162 R3162 16.5 163 R3163 17 164 R3164 36 165 R3165 18.5 166R3166 17.5 167 R3167 11 168 R3168 7.5 169 R3169 5 170 R3170 4.5 171R3172 12 173

Example 11. Alternative Human Hemolysis Assay Using C5 Depleted Sera

Polypeptides listed in Table 6 were tested for functional activity in ared blood cell hemolysis assay using human C5 depleted sera and purifiedhuman C5 rather than complete human sera. To assess activity,antibody-sensitized sheep erythrocytes (Complement Technology, Tyler,Tex.) were plated 2.5×10⁷ cells/well with 1.5% human C5 depleted sera(Complement Technology, Tyler, Tex.) and 0.5 nM purified human C5(Complement Technology, Tyler, Tex.). The antibody-sensitized sheeperythrocytes were centrifuges at 2,090×gravity for 3 minutes and thenresuspended in fresh GVB++(Complement Technology, Tyler, Tex.). Thehuman C5 depleted sera and purified human C5 were rapidly thawed at 37°C. and then stored on ice or wet ice, respectively. The polypeptidestock (10 mM, DMSO) was serially diluted in DMSO in order to obtain 106-fold dilutions and then GVB++ was added to them. 50 μl of eachpolypeptide dilution was combined with 25 μl C5 depleted sera, 25 μlpurified human C5 and 100 μl cells in individual wells of a 96-welltissue culture-treated clear microtitre plate (USA Scientific, Ocala,Fla.) and resuspended by pipetting. The samples were incubated at 37° C.for one hour. At the completion of the incubation, the plates werecentrifuged at 2,090×gravity for 2 minutes. 100 μl of supernatant wastransferred to a new plate and the absorbance was read at 412 nm. Datawas fit with a log-logit formula producing a dose-response curve andIC₅₀.

TABLE 6 Compounds analyzed Compound Avg. SEQ ID Number IC₅₀ (nM) NO.R3171 5.67 172 R3173 2.5 174 R3174 2.3 175 R3176 1.1 177 R3177 12 178R3179 83 180 R3180 29 181 R3181 1496 182 R3182 13 183 R3183 13.25 184R3184 4 185 R3185 12.5 189 R3186 18 187 R3189 81.5 190 R3190 35.33 191R3191 2.5 192 R3192 1.5 193 R3193 24 194 R3194 15.5 195 R3195 62.5 196R3196 3 197 R3197 4 198 R3198 142 199 R3199 112 200 R3200 88.5 201

Example 12. Enzyme Immunoassay to Assess C5 Inhibition

C5 inhibitory activity was assessed by enzyme immunoassay (EIA).Inhibition of the production of C5a and the membrane attack complex(MAC) were measured by MicroVue EIA kits (Quidel Corporation, San Diego,Calif.).

C5a EIA

Supernatant from a human RBC hemolysis assay of R3002 (SEQ ID NO: 3) andR3008 (SEQ ID NO: 9) was diluted 1:50 and assayed by C5a EIA (FIG. 1).Both polypeptides inhibited the formation of C5a. R3002 (SEQ ID NO: 3)had an IC₅₀ of 5.4 nM, R3008 (SEQ ID NO: 9) had an IC₅₀ of 54.5 nM.

Membrane Attack Complex (MAC) EIA

The MAC EIA was performed on the diluted supernatant (1:5) of R3008 (SEQID NO: 9) from a human RBC hemolysis assay (FIG. 2). This polypeptidewas shown to inhibit the formation of the MAC with an IC₅₀ of 33 nM.

Example 13. Characterization of Peptidomimetic Binding by FluorescencePolarization

Fluorescence polarization (FP) allows binding events to be measured in ahomogenous solution (Banks, P. et al., Impact of a red-shifted dye labelfor high throughput fluorescence polarization assays of Gprotein-coupled receptors. J Biomol Screen. 2000 October; 5(5):329-34and Parker, G. J. et al., Development of high throughput screeningassays using fluorescence polarization: nuclear receptor-ligand-bindingand kinase/phosphatase assays. J Biomol Screen. 2000 April; 5(2):77-88).The key concept of FP is that the degree by which a fluorophorepolarizes light is inversely related to its molecular rotation (Lea, W.A. et al., Fluorescence polarization assays in small molecule screening.Expert Opin Drug Discov. 2011 January; 6(1): 17-32), and a fluorophorebound to a much larger target protein rotates more slowly than anunbound fluorophore, resulting in an increase in polarization that canbe quantified. FP has been used increasingly in high throughputcampaigns as a method to measure ligand-target binding (Parker, G. J. etal., Development of high throughput screening assays using fluorescencepolarization: nuclear receptor-ligand-binding and kinase/phosphataseassays. J Biomol Screen. 2000 April; 5(2):77-88), for equilibriumdissociation constant (K_(D)) determination (Prystay, L. et al.,Determination of equilibrium dissociation constants in fluorescencepolarization. J Biomol Screen. 2001 June; 6(3):141-50), and leaddiscovery through competitive binding assays (Tian, W. et al.,Development of novel fluorescence polarization-based assay for studyingthe (3-catenin/Tcf4 interaction. J Biomol Screen. 2012 April;17(4):530-4).

Materials and Methods

FP was used for screening competitive polypeptide inhibitors of the C5protein. Probe R3076 (SEQ ID NO: 40) was generated by incubating theparent polypeptide, R3072 (SEQ ID NO: 20) with BODIPY-TMR-X, SE (LifeTechnologies, Grand Island, N.Y.) in DMF (Sigma, Saint Louis, Mo.) for 4hours. The BODIPY-TMR dye attached to the C-terminal lysine of theprotein and the subsequent labeled probe was purified by HPLC.

The equilibrium dissociation constant (K_(D)) for binding of R3076 (SEQID NO: 40) to human C5 protein (Complement Technology, Tyler, Tex.) wasdetermined by incubating a solution of 25 nM R3076 (SEQ ID NO: 40) withincreasing concentrations of C5 protein. Polarization was measured overtime, until binding reached equilibrium. K_(D) was determined usingGraphpad Prism (using “Saturating Binding Curves, One Site—SpecificBinding With Hill Slope” as the curve fit to determine K_(D)).Equilibrium was reached after 10 minutes, with values for K_(D), hillslope and maximal binding remaining stable over 60 minutes. A finalK_(D) value of 8.07 nM (0.53 standard deviation) was determined byaveraging K_(D) values from 10 to 60 minutes. Based on this information,25 nM and 50 nM concentrations for R3076 (SEQ ID NO: 40) and C5 protein,respectively, were chosen for use in the competition assay. Theseconcentrations represented the approximate level of protein necessaryfor 95% probe binding to C5 protein. R3023 (SEQ ID NO: 104) is ascrambled polypeptide variant of R3002 (SEQ ID NO: 3) and was includedin all assays as a negative control.

Human C5 protein was diluted to 200 nM in assay buffer, composed of TBS(EMD Millipore, Billerica, Mass.)+0.005% Triton-X (Sigma, Saint Louis,Mo.). 10 μl of assay buffer was added to all wells of a black,non-binding, 384-well assay plate (Greiner, Monroe, N.C.) and 10 μl ofdiluted C5 protein stock was added to experimental and designatedcontrol wells.

Probe R3076 (SEQ ID NO: 40) was diluted 1 to 10 in DMSO (LifeTechnologies, Grand Island, N.Y.) and 30 μl of that stock was diluted in3 ml of assay buffer to yield a 100 nM stock. 10 μl of this workingstock was then added to each well in the assay plate. The assay platewas incubated at room temperature, protected from light, for 20 minutesto allow binding to reach equilibrium.

Test articles listed in Table 7 were subsequently diluted in DMSO thenassay buffer, comprising 10 2-fold dilutions and were then added to theassay plate in triplicate, rapidly. The assay plate was then incubatedin the Paradigm (Molecular Devices, Sunnyvale, Calif.) plate reader for60 minutes at 25° C.

After incubation for 60 minutes, the plate was read using the ParadigmFP protocol (Molecular Devices, Sunnyvale, Calif.) and raw polarizationvalues were imported into Graphpad Prism. K_(i)(using the One Site K_(i)Curve fitting model, probe concentration=25 nM, K_(D)=8.07 nM, withbaseline constrained to the average of the 0% binding control) and IC₅₀(log inhibitor vs response, 4 parameter curve fit) were determined inGraphpad.

Results

All test articles were able to compete with the labeled probe forbinding to human C5 protein (FIG. 3, Table 7). R3003 (SEQ ID NO: 4) wasthe most potent polypeptide tested, with a K_(i) value of 9.54 nM. R3023(SEQ ID NO: 104) binding was not detected at the highest concentrationtested.

TABLE 7 Competitive fluorescence polarization data Compound SEQ ID K_(i)nM, K_(i) nM, IC₅₀ nM, IC₅₀ nM, Avg, Std. Dev. Avg, Std. Dev. Number NOExp 1 Exp 2 Exp 1 Exp 2 nM K_(i) K_(i) nM IC₅₀ IC₅₀ R3003 4 10.39 8.6972.65 64.06 9.54 1.20 68.36 6.07 R3011 31 73.17 86.91 294.8 261.3 80.049.72 278.1 23.69 R3014 55 1405 1585 6064 7579 1495 127.3 6822 1071 R3023104 >1000 >1000 >1000 >1000 >1000 — >1000 — R3043 50 866.1 882.1 43324122 874.1 11.31 4227 148.5 R3050 23 71.08 57.6 266.9 292.7 64.34 9.53279.8 18.24

Data shown in Table 7 were obtained from curve fitting analysisperformed by Graphpad Prism software as described above. Triplicatevalues were averaged to yield the data points presented in eachexperiment. Of the polypeptides tested, R3003 (SEQ ID NO: 4) wasidentified originally by mRNA display selection. The affinity of R3003(SEQ ID NO: 4) for C5 was verified by the results of FP analysisdisplaying low K_(i) as well as IC₅₀ values. Inhibitors R3011 (SEQ IDNO: 31) and R3050 (SEQ ID NO: 23) also displayed relatively strongaffinity for C5. A control polypeptide, R3023 (SEQ ID NO: 104),displayed no affinity for C5, while inhibitors R3014 (SEQ ID NO: 55) andR3043 (SEQ ID NO: 50) displayed weak affinity.

Example 14. Analysis of Compound Stability in Plasma

Compounds were assayed for stability in human plasma under the followingconditions. Human plasma was obtained from Bioreclamation (Westbury,N.Y.) and collected in sodium heparin. Plasma was adjusted to pH 7.4.DMSO stocks at 10 mM concentration were prepared for the test compounds.Aliquots of the DMSO solutions were dosed into 1 mL of plasma, which hadbeen pre-warmed to 37° C., at a final test compound concentration of 10M. The vials were kept in a benchtop THERMOMIXER® (Eppendorf, Hauppauge,N.Y.) for the duration of the experiment. Aliquots (100 μL) were takenat each timepoint and added to a 96-well plate that had been pre-filledwith 300 μL of an acetonitrile solution containing mixture of theinternal standards (metoprolol, propranolol and warfarin each at 500ng/mL). Samples were stored at 4° C. until the end of the experiment.After the final timepoint was sampled, the plate was mixed and thencentrifuged at 3,000 rpm for 10 minutes. Aliquots of the supernatantwere removed and analyzed by LC-HRAMS. Liquid chromatography settingsare listed in Table 8 and mass spectrometry settings are listed in Table9.

TABLE 8 Liquid chromatography settings Column: Luna C18 (Luna, Torrance,CA) 50 mm × 2.0 mm, 3 μM M.P. Buffer: Aqueous Reservoir (A): 0.1% Aceticacid in water Organic Reservoir (B): 0.1% Acetic acid in MeOH:MeCN = 1:1Time Flow rate (Min) (mL/min) % A % B Gradient Program: 0.0 0.3 100 0 50.3 0 100 7.5 0.3 0 100 7.6 0.45 100 0 10.5 0.3 100 0 Total Run Time:10.5 minutes Autosampler: Agilent 1100 Bin (Agilent, Santa Clara, CA)Injection loop 20 μL volume: Injection volume: 10 μL AutosamplerMethanol/water 1:1; with 0.2% formic acid Wash 1: AutosamplerMethanol/2-propanol: 1/1; with 0.2% formic acid Wash 2:

TABLE 9 Mass spectrometry settings Instrument: LTQ Orbitrap XL (ThermoScientific, St. Louis, MO) Positive Mode: Electrospray, positive mode(+5000 V) Interface: High Resolution Mass Spectroscopy Mode: CapillaryTemperature: 275° C. Ion Source Capillary Voltage: 47 Settings: Sheathgas: 45 Auxiliary gas: 15 Sweep gas: 10 Orbitrap Scan Range 200-2000,Resolution = 30000 Settings: (Full width at half maximum) Setting forMS/MS Data Dependent Acquisition Isolation Width: 2 Normalized CollisionEnergy: 35

The concentration of test compound R3050 (SEQ ID NO: 23) was determinedby comparison to a previously determined calibration curve (Table 10).

TABLE 10 Stability profile Percent remaining at time (hrs) Half-Life 0hrs 2 hrs 4 hrs 12 hrs 24 hrs (min.) 100 108.9 100.4 88 98.1 >1440

Under these conditions, R3050 (SEQ ID NO: 23) was shown to be highlystable.

Example 15. Polypeptide Variants Comprising Tryptophan Analogs

In some embodiments, polypeptides of the present invention comprise7-azatryptophan. To determine the importance of this residue in C5inhibition, amino acid substitution analysis was carried out wherein7-azatryptophan was replaced by natural tryptophan as well as variousother tryptophan analogs including 5-fluorotryptophan [(5-F)W],1-methyl-tryptophan [(1-Me)W], D-tryptophan and 5-methyl-O-tryptophan[(5-MeO)W]. Similar polypeptides with non-tryptophan substitutions wereanalyzed as well.

Polypeptide variants of R3002 (SEQ ID NO: 3) and R3008 (SEQ ID NO: 9)were synthesized and tested for their ability to inhibit red blood celllysis as described in Example 10 (see Tables 11 and 12). Of the variantstested, all with substitution of the 7-azatryptophan residuedemonstrated a decreased ability to inhibit red blood cell lysis asindicated by increasing average IC₅₀ values (a measure of the halfmaximal inhibitory concentration, a value used to indicate the amount ofthe inhibitor needed to reduce a given reaction or process by half).

TABLE 117-azatryptophan variant polypeptides of R3002 (SEQ ID NO: 3) analyzed byhuman hemolysis assay Compound Avg. IC₅₀ SEQ Number Sequence (nM) ID NOR3002 Ac-Nvl-C-Y-E-N-Tbg-Y-azaTrp-E-Y-P-Phg-Nvl-NH2       11.9 3 R3041Ac-Y-E-N-Tbg-Y-W-E-Y-P-Phg-Nvl-NH2       71.3 26 R3094Ac-Y-E-N-Tbg-Y-(5-MeO)W-E-Y-P-Phg-Nvl-NH2 >100,000 121 R3110Ac-Y-E-N-Tbg-Y-(1-Me)W-E-Y-P-Phg-Nvl-NH2    2,940 59 R3118Ac-Y-E-N-Tbg-Y-(D-Trp)-E-Y-P-Phg-Nvl-NH2 >100,000 127 R3119Ac-Y-E-N-Y-(D-Trp)-E-Y-P-Phg-Nvl-NH2 >100,000 128 R3128Ac-Nvl-C-Y-N-N-Tbg-E-C-E-Y-P-Phg-Tbg-NH2 >100,000 132 R3067Ac-Nvl-S-Y-E-N-Tbg-Y-A-E-Y-P-Chg-Nvl-NH2  >10,000 76 R3116Ac-Nvl-Nvl-Y-E-N-Tbg-Y-(N-Me)W-E-Y-P-Chg-Nvl-      441 45 NH2 R3017[mXylyl(2,8)]Ac-Nvl-C-Y-E-N-Tbg-Y-C-E-Y-P-Phg- >100,000 103 Nvl-NH2R3129 [mXylyl(2,8)]Ac-Nvl-C-Y-N-N-Tbg-E-C-E-Y-P-Phg- >100,000 133Tbg-NH2

TABLE 127-azatryptophan variant polypeptides of R3008 (SEQ ID NO: 9) analyzed byhuman hemolysis assay Compound Avg. IC₅₀ SEQ Number Sequence (nM) ID NOR3008 [mXylyl(2,10)]Ac-Nvl-C-Phg-T-azaTrp-E-Y-(N-Me)S-       97 9H-C-Nvl-P-Nvl-NH2 R3088[mXylyl(2,10)]Ac-Nvl-C-Phg-T-(5-F)W-E-Y-(N-Me)S- >100,000 118A-C-Nvl-NH2 R3091 [mXylyl(2,10)]Ac-Nvl-C-Phg-T-W-E-Y-(N-Me)S-A-C-     483 46 Nvl-NH2 R3092[mXylyl(2,10)]Ac-Nvl-C-Phg-T-F-E-Y-(N-Me)S-A-C-  >50,000 92 Nvl-NH2R3109 [mXylyl(2,10)]Ac-Nvl-C-Phg-T-(1-Me)W-E-Y-(N- >100,000 126Me)S-A-C-Nvl-NH2 R3131[mXylyl(2,10)]Ac-Nvl-C-Phg-T-W-Asp (T)-Y-(N- >100,000 135Me)S-H-C-Nvl-NH2 R3034 [mXylyl(2,10)]Ac-Nvl-C-Phg-T-A-E-Y-(N-Me)S-H-C- >50,000 85 Nvl-NH2 R3132[mXylyl(2,10)]Ac-Nvl-C-Phg-T-(D-Trp)-E-Y-(N-Me)S- >100,000 136H-C-Nvl-NH2

Example 16. Effect of Polypeptide Truncation and Amino Acid DeletionAnalyzed by Human Hemolysis Assay

C-terminally truncated polypeptide variants of R3021 (SEQ ID NO: 11)were synthesized and assayed by the human hemolysis assay described inExample 10 for their ability to inhibit C5-dependent red blood celllysis. Average IC₅₀ values (a measure of the half maximal inhibitoryconcentration, a value used to indicate the amount of the inhibitorneeded to reduce a given reaction or process by half) for eachpolypeptide tested are listed in Table 13. Truncated polypeptidesdemonstrated decreased ability (as indicated by an increase in the IC₅₀value) to inhibit red blood cell lysis with those variants lackingtryptophan having the largest IC₅₀ values.

Additionally, polypeptide variants of R3021 (SEQ ID NO: 11) withinternal amino acid deletions were synthesized and assayed (see Table14) for their ability to inhibit C5-dependent red blood cell lysisaccording to the method described in Example 10. Additionally, theN-terminal methionine in these variants was replaced by an acetyl group.Average IC₅₀ values for each polypeptide tested are listed in Table 14.Interestingly, acetyl group replacement of the N-terminal methioninealone [R3048 (SEQ ID NO: 19)] increased the ability of the polypeptideto inhibit red blood cell lysis. Removal of internal residue D [R3124(SEQ ID NO: 130)] or internal residues DVY [R3125 (SEQ ID NO: 131),corresponding to residues 8, 9 and 10 from R3021 (SEQ ID NO: 11)] led toa decrease in the ability of the polypeptide to inhibit red blood celllysis.

TABLE 13C-terminally truncated polypeptide variants of R3021 (SEQ ID NO: 11) analyzedby human hemolysis assay Compound Avg. IC₅₀ SEQ Number Sequence (nM)ID NO R3021 [mXylyl(2,7)]M-C-V-E-R-F-C-D-V-Y-W-E-F-NH2       27.5 11R3043 [mXylyl(2,7)]M-C-V-E-R-F-C-D-V-Y-W-E-NH2      934 50 R3044[mXylyl(2,7)]M-C-V-E-R-F-C-D-V-Y-W-NH2  >50,000 88 R3045[mXylyl(2,7)]M-C-V-E-R-F-C-D-V-Y-NH2 >100,000 107 R3046[mXylyl(2,7)]M-C-V-E-R-F-C-D-V-NH2 >100,000 108 R3047[mXylyl(2,7)]M-C-V-E-R-F-C-NH2 >100,000 109

TABLE 14Polypeptide variants of R3021 (SEQ ID NO: 11) with internal amino aciddeletions analyzed by human hemolysis assay Compound Avg. IC₅₀ SEQNumber Sequence (nM) ID NO R3021[mXylyl(2,7)]M-C-V-E-R-F-C-D-V-Y-W-E-F-NH2       27.5 11 R3048[mXylyl(1,6)]Ac-C-V-E-R-F-C-D-V-Y-W-E-F-NH2       19.3 19 R3124[mXylyl(1,6)]Ac-C-V-E-R-F-C-V-Y-W-E-F-NH2 >100,000 130 R3125[mXylyl(1,6)]Ac-C-V-E-R-F-C-W-E-F-NH2 >100,000 131

Example 17. Incorporation of Albumin-Binding Polypeptides

Polypeptides are conjugated to one or more polypeptides that modulateplasma protein binding. These polypeptides, referred to herein as“albumin-binding polypeptides” are listed in Table 15.

TABLE 15 Albumin-binding polypeptides SEQ IDAlbumin-binding polypeptide sequence NOAc-R-L-I-E-D-I-C-L-I-P-R-W-G-C-L-W-E-D-D-NH2 202Q-R-L-M-E-D-I-C-L-P-R-W-G-C-L-W-E-D-D-F-NH2 203Ac-Q-R-L-I-E-D-I-C-L-P-R-W-G-C-L-W-E-D-D-F-NH2 204

Albumin-binding polypeptides are cyclized by disulfide bond formation atcysteine residues. In some embodiments, albumin-binding polypeptides areconjugated by either their N or C-terminal ends, thus having slightlydifferent structures (e.g. no acetyl group).

Example 18. Incorporation of Cell Penetrating Polypeptides

Polypeptides are conjugated to a polypeptide that has cell penetratingproperties. These polypeptides are listed in Table 16 and are describedin Milletti, F., Cell-penetrating polypeptides: classes, origin, andcurrent landscape. Drug Discov Today. 2012 August; 17(15-16):850-60.

TABLE 16 Cell penetrating polypeptides Cell penetrating polypeptideSEQ ID NO R-K-K-R-R-R-E-S-R-K-K-R-R-R-E-S 205 R-K-K-R-R-Q-R-R-R 206R-Q-I-K-I-W-F-Q-N-R-R-M-K-W-K-K 207 A-A-V-L-L-P-V-L-L-A-A-P 208V-P-T-L-K 209 P-L-I-L-L-R-L-L-R-G-Q-F 210

Example 19. Analysis of Polypeptide Mixtures Comprising Amino AcidStereoisomers

Polypeptides R3136 (SEQ ID NO: 137) and R3137 (SEQ ID NO: 138) weresynthesized according to the amino acid sequences of R3085 (SEQ ID NO:90) and R3082 (SEQ ID NO: 116), respectively with the exception of thereplacement of Phg in each with D-Phg (see Table 17). Compositionscomprising either R3136 (SEQ ID NO: 137) and R3085 (SEQ ID NO: 90) orR3137 (SEQ ID NO: 138) and R3082 (SEQ ID NO: 116) were analyzed fortheir ability to inhibit red blood cell lysis according to the humanhemolysis assay described in Example 10. The composition comprisingR3136 (SEQ ID NO: 137) and R3085 (SEQ ID NO: 90) yielded an average IC₅₀(nM) of >50,000, while the composition comprising R3137 (SEQ ID NO: 138)and R3082 (SEQ ID NO: 116) yielded an average IC₅₀ (nM) of >100,000.

TABLE 17 Compounds used in amino acid stereoisomer polypeptide mixturesSEQ Compound ID Number Sequence NO. R3136[mXylyl(2,10)]heptanoyl-Nvl-C-(D-Phg)-T-azaTrp-E-Y-(N-Me)S-A-C-Nvl- 137NH2 R3137[mXylyl(1,9)]heptanoyl-C-(D-Phg)-T-azaTrp-E-Y-(N-Me)S-A-C-Nvl-NH2 138R3085 [mXylyl(2,10)]heptanoyl-Nvl-C-Phg-T-azaTrp-E-Y-(N-Me)S-A-C-Nvl-NH290 R3082 [mXylyl(1,9)]heptanoyl-C-Phg-T-azaTrp-E-Y-(N-Me)S-A-C-Nvl-NH2116

Example 20. Pharmacokinetic Studies in Non-Human Primates

Pharmacokinetic studies were carried out in non-human primates using thecompounds listed in Table 18. In the table, “Cmpd” refers to compoundand “Avg” refers to average.

TABLE 18 Compounds tested in in vivo studies SEQ Cmpd Avg ID No.Sequence IC₅₀ NO. R3152[mXylyl(1,6)]Ac-C-V-E-R-F-C-D-Tbg-Y-azaTrp-E-Y-P-Chg-Nvl-NH2 16.4 153R3201 [mXylyl(1,6)]Ac-C-V-E-R-F-C-D-Tbg-Y-azaTrp-E-Y-P-Chg-Nvl 7.7 211

The plasma concentration of polypeptide R3152 (SEQ ID NO: 153) wasdetermined in cynomolgus monkeys following a single intravenous (IV)dose. Three male animals received 3 mg/kg polypeptide and plasmaconcentrations of polypeptide were determined using LC/MS-MS followingacetonitrile precipitation and extraction on Sirocco ProteinPrecipitation plate (Waters Corporation, Milford, Mass.) Pharmacokinetic(PK) parameters were calculated from the time course (see FIG. 4) of thecombined plasma concentrations of R3152 (SEQ ID NO: 153,) determinedimmediately post-dose to up to 48 h post-dose. Plasma drug levels fellrapidly during an initial distribution phase (<1 hour) and thenplateaued and were detectable for up to 48 hours. R3152 (SEQ ID NO: 153)had a mean terminal half-life of 10.9±0.8 hours. The mean clearance ratewas 0.129±0.0122 L/hr/kg which is approximately 5% of the liver bloodflow of a typical monkey (2.6 L/hr/kg). The mean volume of distributionwas 1.49±0.152 L/kg which is approximately double the total body waterfor a typical monkey (0.7 L/kg). The average AUC∞ was 23319±2120hr*ng/mL.

R3152 (SEQ ID NO: 153) binds with high affinity to primate C5 proteinand blocks the complement pathway by preventing the generation of theC5a and C5b products and the production of a multimeric Membrane AttackComplex (MAC). The inhibition of complement-mediated MAC formation inplasma samples from the above PK study was examined using an establishedex vivo assay (see the human hemolysis assay described in Example 10),in which plasma was diluted 1:100 and incubated with activated sheep redblood cells (Complement Technology, Tyler, Tex.). At each time-point,the hemolytic activity was determined as an indicator of active serumcomplement (see FIG. 4). In plasma containing >200 ng/mL R3152 (SEQ IDNO: 153) there was a clear inhibition of complement-mediated hemolysis,indicating a blockade of MAC formation. Exogenous R3152 (SEQ ID NO: 153)added to normal cynomolgus plasma has an IC₅₀=2-20 ng/mL. Hemolyticactivity returned to normal levels 48 hours after dosing as plasmalevels of R3152 (SEQ ID NO: 153) fell below 100 ng/ml.

Example 21. Pharmacokinetic Studies in Rat

R3152 (SEQ ID NO: 153) was delivered as an intravenous (IV) orsubcutaneous (SC) dose to male rats at 2 and 30 mg/kg, respectively.Following IV dosing, R3152 (SEQ ID NO: 153) was monitored by usingLC/MS-MS following acetonitrile precipitation and extraction on SiroccoProtein Precipitation plate (Waters Corporation, Milford, Mass.) asdescribed above. Pharmacokinetic (PK) parameters were calculated fromthe time course of the combined plasma concentrations of R3152 (SEQ IDNO: 153) and its equipotent C-terminally deamidated metabolite, R3201(SEQ ID NO: 211). Results are presented in FIG. 5. In the figure,circles represent concentrations obtained after SC dose and squaresrepresent concentrations obtained after IV dose.

R3152 (SEQ ID NO: 153)/R3201 (SEQ ID NO: 211) exhibited a fastdistribution phase, followed by a slow elimination with a t_(1/2)=5.3hrs. A similar elimination rate was observed after SC dosing of 30mg/kg, with approximately 65% bioavailability of dose, based on AUC. TheT_(max) of 4 hrs and prolonged drug exposure seen in SC doses allowedfor extended coverage of the therapeutic concentration in plasma. AsR3152 (SEQ ID NO: 153) and R3201 (SEQ ID NO: 211) do not bind to rat C5,very little inhibitory activity was observed in ex vivo hemolysisassays.

Lipidated and non-lipidated compounds R3183 (SEQ ID NO: 184) and R3176(SEQ ID NO: 177), respectively, were evaluated for pharmacokineticproperties in Male Sprague-Dawley Rats following intravenous orsubcutaneous administration. FIGS. 6A and 6B show the results. FIG. 6Ashows Male Sprague-Dawley rats (n=3) injected intravenously with asingle 2 mg/kg dose. Blood samples were collected at indicated timepoints, processed into plasma, and analyzed for the indicated compoundby LC-MS. Black circles: R3176 (SEQ ID NO: 177) (unlipidated compound);Open circles: R3183 (SEQ ID NO: 184) (C16 lipidated compound). FIG. 6Bshows Male Sprague-Dawley rats (n=3) injected subcutaneously with asingle 15 mg/kg dose. Blood samples were collected at indicated timepoints, processed into plasma, and analyzed for the indicated compoundby LC-MS. Black circles: R3176 (SEQ ID NO: 177) (unlipidated compound);Open circles: R3183 (SEQ ID NO: 184) (C16 lipidated compound).Lipidation resulted in an increase in exposure as assessed bydetermination of the Area under the curve (AUC) by 2.1-fold by theintravenous route and 2.7-fold by the subcutaneous route.

Example 22. Inhibition of Hemolysis in the Thrombin-Induced ComplementPathway

Thrombin can induce complement activity by cleaving C5 into C5_(T) whichwill then be cleaved into C5a and C5b_(T). C5b_(T), like C5b, willassociate with C6 and the remaining terminal components of thecomplement pathway, C7, C8 and C9, which will lead to formation of theMembrane Attack Complex (MAC) causing lysis of red blood cells(Krisinger, et al., (2014). Blood. 120(8):1717-1725). Thus, R3183 and ananti-C5 monoclonal antibody similar to ECULIZUMAB® were tested for theirability to inhibit hemolysis through the thrombin-induced complementpathway.

To assess inhibitor activity, C5 (Complement Technology, Tyler, Tex.)was added to achieve a concentration of 400 nM, and the sample wasincubated with C6 at a final concentration of 600 nM (ComplementTechnology, Tyler, Tex.) and thrombin at a concentration of 50 nM(Enzyme Research Laborites, South Bend, Ind.) at 37° C. for 30 minutes,in the presence of either R3183 or the anti-C5 monoclonal antibodysimilar to ECULIZUMAB®, or no inhibitor. The reaction was stopped withthe addition of hirudin to 150 nM (Cell Sciences, Canton, Mass.) in thebuffer GVB+EDTA (Complement Technology, Tyler, Tex.) and incubated 5minute at room temperature. These diluted samples were mixed withantibody-sensitized sheep erythrocytes (Complement Technology, Tyler,Tex.) in a 96-well microtitre plate (USA Scientific, Ocala, Fla.) andincubated at 37° C. for 5 minutes. C7 (Complement Technology, Tyler,Tex.) was then added to the wells to achieve a concentration of 15 nMand the plate was returned to 37° C. for 15 minutes. A complex of C8 (10nM; Complement Technology, Tyler, Tex.) and C9 (25 nM; ComplementTechnology, Tyler, Tex.) was then added to the assay mixture and thesamples were incubated for 30 minutes at 37° C. Following incubation,the plate was centrifuged at 1000×g and 100 μL of supernatant wastransferred to a new microtitre plate and the absorbance was read at 412nm. The resulting data are shown in FIG. 7. R3183 was found to inhibithemolysis by the thrombin-induced complement pathway at concentrationshigher than 6 ng/mL, while the anti-C5 monoclonal antibody did not.

Example 23. Surface Plasmon Resonance Analysis of R3183 Binding

Surface Plasmon Resonance (SPR) experiments were conducted at 25° C.using the ProteOn XPR36 system from BioRad Laboratories, Inc. (Hercules,Calif.). C5 protein [or human serum albumin (HSA) control] wasimmobilized by direct amine coupling on a ProteOn GLH sensor chipdesigned for maximal binding capacity using pH 5 acetate buffer. Kineticcharacterization of R3183 binding was performed in binding buffercontaining 10 mM HEPES, pH 7.4, 150 mM NaCl, 0.5 mM MgCl₂, 0.15 mMCaCl₂, 0.005% Tween-20, and 1% DMSO to determine k_(on), k_(off), andK_(D). Data analysis was performed using BioRad ProteOn Managersoftware. Sensograms were fit to the heterogeneous ligand model (seeFIG. 8). The concentrations of R3183 evaluated in this experiment were3.3, 1.1, 0.37 and 0.12 μM as indicated in the figure.

R3183 was found to have a k_(a) (1/Ms) of 1.18×10⁵ for C5, as well as ak_(d) (1/s) of 3.04×10⁻⁴ and a K_(D) (M) of 2.58×10⁻⁹. Values for HSAbinding were: k_(a) (1/Ms) of 3.01×10⁴, k_(d) (1/s) of 1.76×10⁻¹ and aK_(D) (M) of 5.86×10⁻⁶.

Example 24. Structural Studies

Crystallography studies were carried out to compare C5 binding sitesbetween a close chemical analog of R3183 and ECULIZUMAB® (AlexionPharmaceuticals, Cheshire, CT). An induced fit model revealed that theanalog binds to a site distinct from that reported for ECULIZUMAB®.

Example 25. Immunoassay Analysis of R3183 Inhibitory Activity

C5 inhibitory activity for R3183 was assessed by enzyme immunoassay(EIA). Inhibition of the production of C5a and the membrane attackcomplex (MAC) were measured by MicroVue EIA kits (Quidel Corporation,San Diego, Calif.).

Supernatant from a human red blood cell (RBC) hemolysis assay of R3183was diluted 1:50 and assayed by C5a EIA (FIG. 9). R3183 inhibited theformation of C5a with an IC₅₀ of 11 nM.

The MAC EIA was performed with R3183 on the same supernatant, diluted1:5 (see FIG. 10). This compound was shown to inhibit the formation ofthe MAC with an IC₅₀ of 6.9 nM. Serum samples containing differentconcentrations of R3183 were also analyzed for inhibition of thealternative pathway of complement activation using the WIESLAB®complement system screening kit (Euro Diagnostica, Malmo, Sweden).Results indicated an IC₅₀ of 12.5 nM for R3183.

Example 26. Human Hemolysis Assay

R3183 inhibitor activity was compared to an ECULIZUMAB®-like antibody(mAb-C5) using a red blood cell hemolysis assay. A DNA expression vectorfor a IgG monoclonal antibody inhibitor of C5 (mAb-C5) was constructedfrom the published sequence for the variable heavy and light chainsequences of h5G1.1, h5G1.1VHC+F and h5G1.1VLC+F, respectively (Thomaset al. 1996. Molecular Immunology. 33(17-18):1389-401). The humanconstant light chain kappa and IgG2 constant heavy chain sequences wereused for the constants regions of the antibody. This antibody wasexpressed from human embryonic kidney cells (HEK293) and purified byProtein A affinity chromatography. Antibody-sensitized sheeperythrocytes (Complement Technology, Tyler, Tex.) were plated at 2.5×10⁷cells/well with complete human sera (Complement Technology, Tyler Tex.)with or without inhibitors to determine the inhibitory effect of thecompounds on the lysis of red blood cells. Cells were centrifuged for 3minutes at 2,090×gravity and resuspended in fresh GVB++ buffer(Complement Technology, Tyler Tex.). Human sera was rapidly thawed at37° C. and then stored on ice until diluted into GVB++. Ten 6-foldserial dilutions of each compound (10 mM stock, DMSO) were performed inDMSO and then added to buffer. 50 μl of each compound dilution wascombined with sera and 100 μl of cells in individual wells of a 96-welltissue culture-treated clear microtitre plate (USA Scientific, Ocala,Fla.) and resuspended by pipetting. Samples were incubated at 37° C. forone hour. Following incubation, plates were centrifuged at 2,090×gravityfor 2 minutes. 100 μl of supernatant was transferred to a new plate andthe absorbance was read at 412 nm. Data was fit with a log-logit formulaproducing a dose-response curve and IC₅₀ (see FIG. 11A). The IC₅₀ forR3183 was 8.1 nM as compared to 0.2 nM for mAb-C5. Interestingly, themolecular weight for R3183 is 2 kDa, while the molecular weight formAb-C5 is 140 kDa, indicating a much lower overall amount needed fortherapeutic applications.

Similar studies indicated that R3183 is also active in serum fromcynomolgus monkeys and pigs, but less active in the serum of rodents anddogs. When 1% human serum was compared to 1% cynomolgus monkey serum inthe assay, R3183 had an IC₅₀ of 1.9 nM and an IC₉₀ of 5.2 nM with humanserum as compared to an IC₅₀ of 4.2 nM and an IC₉₀ of 13.2 nM withcynomolgus monkey serum (see FIG. 11B).

Example 27. Pharmacodynamic and Pharmacokinetic Studies

Studies were carried out in non-human primates to determine the dose andregimen of R3183 needed to achieve sustained complement inhibition, aswell to determine the pharmacokinetic profile of R3183 during theadministration period and over time after administration of the finaldose. A low dose (Dose A) group of three male animals received 0.3 mg/kgof R3183 daily for seven days and were monitored for 10 days thereafter.A second, high dose group of three male animals received a single lowdose (Dose A; 0.3 mg/kg) of R3183 on day 1 and received higher dailydoses (Dose B; 3.0 mg/kg) of R3183 for 6 days thereafter. This group wasalso monitored for 10 days after receiving the final dose. Plasmasamples were obtained prior to each dose, immediately post-dose andtwice thereafter each day until daily dosing was completed. Samples weretaken periodically thereafter for 10 days. Concentrations of R3183 weredetermined in each plasma sample using LC/MS-MS following acetonitrileprecipitation and extraction on Sirocco Protein Precipitation plates(Waters Corporation, Milford, Mass.).

R3183 binds with high affinity to primate C5 protein and blocks thecomplement pathway by preventing the generation of the C5a and C5bproducts and the production of a multimeric Membrane Attack Complex(MAC). The inhibition of complement-mediated MAC formation in the sameplasma samples was examined using an established ex vivo assay in whichplasma was diluted 1:100 and incubated with activated sheep red bloodcells (Complement Technology, Tyler, Tex.). At each time-point, thehemolytic activity was determined as an indicator of active serumcomplement (see FIGS. 12A and 12B). In the low dose group, 90%inhibition was observed with some R3183 plasma concentrations between2000 and 4000 ng/ml, with greater than 90% inhibition with mostconcentrations above 4000 ng/ml (FIG. 12A). R3183 inhibition ofhemolytic activity was detected up to 10 days after final treatments. Acombined graphical representation of the data from low and high dosegroups is presented in FIG. 13.

Example 28. Inhibition of Lysis with Addition of Terminal Components

C5 was incubated with C6 and thrombin at 37° C. for 30 minutes with andwithout R3183. The reaction was stopped with the addition of hirudin.The diluted samples were mixed with antibody-sensitized sheeperythrocytes and then incubated for 5 minutes at 37° C. Purified C7 wasadded and incubated for 15 minutes at 37° C. A complex of purified C8and C9 was then added and the mixture was incubated for 30 minutes at37° C. The plate was centrifuged and the supernatant transferred andread at 412 nm. Results demonstrate strong inhibition in the presence ofR3183 (see FIG. 14).

Example 29. Hemolysis Analysis in PNH Patient Samples

Activation and inhibition of the alternative complement pathway in bloodcells obtained from patients with paroxysmal nocturnal hemoglobinuria(PNH) was monitored in the presence or absence of complement inhibitorsby flow cytometry as described in Risitano et al., 2012. Blood. 119(6):6307-16, the contents of which are herein incorporated by reference intheir entirety. Complement activation may be increased when serum isacidified. Here, red blood cells from patients having PNH were incubatedin serum in the presence or absence of R3144 (SEQ ID NO: 145) orECULIZUMAB®. Heat inactivated serum was used as a negative control(i.e., no red blood lysis takes place). The pH in samples was lowered byaddition of hydrochloric acid (1:20 dilution of 0.1 N HCl) to initiatecomplement activation. Samples were incubated for 24 hours at 37° C.before analysis. Red blood cells were pelleted, and 1 μl was resuspendedin 1 ml saline. Samples were then incubated with anti-CD59 antibodieswith phycoerythrin labels as well as fluorescein isothiocyanate(FITC)-labeled anti-C3 antibodies for 1 hour prior tofluorescence-associated cell sorting (FACS) analysis. Results indicatedsimilar levels of inhibition above baseline levels for both R3144 andECULIZUMAB®.

What is claimed is:
 1. A method of reducing hemolysis in a subject, themethod comprising administration of a C5 inhibitor to the subject,wherein the C5 inhibitor is administered at a frequency of from aboutevery 12 hours to about every 72 hours, and wherein hemolysis in subjectplasma is reduced by at least 90% during the course of administration.2. The method of claim 1, wherein the C5 inhibitor is administereddaily.
 3. The method of claim 1, wherein the C5 inhibitor is apolypeptide.
 4. The method of claim 3, wherein the polypeptide comprisesfrom about 10 to about 18 amino acids.
 5. The method of claim 3, whereinthe polypeptide comprises a cyclic loop.
 6. The method of claim 3,wherein the polypeptide comprises an amino acid sequence with at least80% sequence identity to the amino acid sequence of SEQ ID NO:
 192. 7.The method of claim 6, wherein the polypeptide comprises a C-terminallysine, wherein the C-terminal lysine is conjugated with a lipid.
 8. Themethod of claim 7, wherein the polypeptide is conjugated with ahydrophilic polymer.
 9. The method of claim 8, wherein the hydrophilicpolymer comprises polyethylene glycol.
 10. The method of claim 7,wherein the polypeptide comprises SEQ ID NO:
 184. 11. The method ofclaim 7, wherein the polypeptide comprises SEQ ID NO:
 194. 12. Themethod of claim 1, wherein the C5 inhibitor is administered at a dose offrom about 0.01 mg/kg to about 20 mg/kg.
 13. The method of claim 12,wherein the C5 inhibitor is administered at a dose of from about 0.3mg/kg to about 3 mg/kg.
 14. The method of claim 1, wherein the C5inhibitor is administered at a dose sufficient to achieve C5 inhibitorplasma levels in the subject of from about 2 μg/ml to about 20 μg/ml.15. The method of claim 14, wherein the C5 inhibitor is administered ata dose sufficient to achieve C5 inhibitor plasma levels in the subjectof about 4 μg/ml.
 16. The method of claim 1, wherein the subject has adisease, disorder, or condition wherein C5 cleavage leads to progressionof the disease, disorder, or condition.
 17. The method of claim 16,wherein the disease, disorder, or condition comprises paroxysmalnocturnal hemoglobinuria (PNH), atypical hemolytic uremic syndrome, ormyasthenia gravis.
 18. The method of claim 1, wherein the subject hasPNH.
 19. The method of claim 1, wherein the subject has previously beentreated with eculizumab.
 20. The method of claim 19, wherein the subjectis also receiving treatment with eculizumab.
 21. The method of claim 19,wherein treatment with eculizumab is ineffective.
 22. The method ofclaim 21, wherein the subject has an eculizumab-resistant C5polymorphism.